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Surgery_Schwartz_3402
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against particular transcription fac-tor (TF), DNA sequences bound by this TF are pulled down CellisolationPrimaryculturePropagationTissuesampleProduction of recombinant proteinsAnalysis of gene functionTransfectionwith DNAABFigure 15-21. Cell culture and transfection. A. Primary cells can be isolated from tissues and cultured in medium for a limited period of time. After genetic manipulations to over-come the cell aging process, primary cells can be immortalized into cell lines for long-term culture. B. DNA can be introduced into cells to produce recombinant gene products or to ana-lyze the biologic functions of the gene.and examined. The TF binding consensus sequences is then pre-dicted, and if the TF binds to a promoter region, it is likely that the gene using this promoter is regulated by this TF. If using an antibody against an epigenetic modification, the modified regions are marked up into the genome to facilitate identifica-tion of potential epigenetic regulating mechanisms.By
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Surgery_Schwartz. against particular transcription fac-tor (TF), DNA sequences bound by this TF are pulled down CellisolationPrimaryculturePropagationTissuesampleProduction of recombinant proteinsAnalysis of gene functionTransfectionwith DNAABFigure 15-21. Cell culture and transfection. A. Primary cells can be isolated from tissues and cultured in medium for a limited period of time. After genetic manipulations to over-come the cell aging process, primary cells can be immortalized into cell lines for long-term culture. B. DNA can be introduced into cells to produce recombinant gene products or to ana-lyze the biologic functions of the gene.and examined. The TF binding consensus sequences is then pre-dicted, and if the TF binds to a promoter region, it is likely that the gene using this promoter is regulated by this TF. If using an antibody against an epigenetic modification, the modified regions are marked up into the genome to facilitate identifica-tion of potential epigenetic regulating mechanisms.By
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Surgery_Schwartz_3403
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Surgery_Schwartz
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by this TF. If using an antibody against an epigenetic modification, the modified regions are marked up into the genome to facilitate identifica-tion of potential epigenetic regulating mechanisms.By using next-generation sequencing technology, any potential mutations in a patient can be scrutinized as well as any defects in epigenetic modification. By combining data from different kinds of sequencing (DNA-seq, RNA-seq, ChIP-seq), better understanding of mutation or transcription-caused diseases aligning with epigenetic regulation can be achieved, which will greatly facilitate the diagnosis of patients and per-sonalization of medicine in a fast and economic way by prevent-ing unnecessary medical costs and procedures.Third-generation sequencing has emerged rapidly at the research level to involve single molecule real-time sequencing (SMRT). Although first developed and marketed by Pacific Biosciences (Pac Bio), Roche is now leading this technology. Third-generation sequencing allows
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Surgery_Schwartz. by this TF. If using an antibody against an epigenetic modification, the modified regions are marked up into the genome to facilitate identifica-tion of potential epigenetic regulating mechanisms.By using next-generation sequencing technology, any potential mutations in a patient can be scrutinized as well as any defects in epigenetic modification. By combining data from different kinds of sequencing (DNA-seq, RNA-seq, ChIP-seq), better understanding of mutation or transcription-caused diseases aligning with epigenetic regulation can be achieved, which will greatly facilitate the diagnosis of patients and per-sonalization of medicine in a fast and economic way by prevent-ing unnecessary medical costs and procedures.Third-generation sequencing has emerged rapidly at the research level to involve single molecule real-time sequencing (SMRT). Although first developed and marketed by Pacific Biosciences (Pac Bio), Roche is now leading this technology. Third-generation sequencing allows
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Surgery_Schwartz_3404
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Surgery_Schwartz
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involve single molecule real-time sequencing (SMRT). Although first developed and marketed by Pacific Biosciences (Pac Bio), Roche is now leading this technology. Third-generation sequencing allows amplification-free single-molecule sequencing with read length extension up to mega-bases and reduced sequencing coverage bias. It can be used to build the gap in the human genome (for example, low complex-ity regions), provide access to structural genomic variants, and simultaneously analyze genome-wide single-nucleotide methyl-ation. So far, clinical application and research is heavily depen-dent on NGS, especially standardizing and reducing the cost of post-NGS analysis. Third-generation sequencing is under fast development and has been adapted to aid and append NGS.Cell ManipulationsCell Culture. Cell culture has become one of the most power-ful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and
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Surgery_Schwartz. involve single molecule real-time sequencing (SMRT). Although first developed and marketed by Pacific Biosciences (Pac Bio), Roche is now leading this technology. Third-generation sequencing allows amplification-free single-molecule sequencing with read length extension up to mega-bases and reduced sequencing coverage bias. It can be used to build the gap in the human genome (for example, low complex-ity regions), provide access to structural genomic variants, and simultaneously analyze genome-wide single-nucleotide methyl-ation. So far, clinical application and research is heavily depen-dent on NGS, especially standardizing and reducing the cost of post-NGS analysis. Third-generation sequencing is under fast development and has been adapted to aid and append NGS.Cell ManipulationsCell Culture. Cell culture has become one of the most power-ful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and
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Surgery_Schwartz_3405
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Surgery_Schwartz
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culture has become one of the most power-ful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and cellular biology.32 Through their ability to be maintained in vitro, cells can be manipulated by the introduc-tion of genes of interest (cell transfection) and be transferred into in vivo biologic receivers (cell transplantation) to study the biologic effect of the interested genes (Fig. 15-21). In common Brunicardi_Ch15_p0479-p0510.indd 49918/02/19 11:12 AM 500BASIC CONSIDERATIONSPART Ilaboratory settings, cells are cultured either as a monolayer (in which cells grow as one layer on culture dishes), considered 2-D, or in suspension or biomedical material skeleton such as hydrogel, considered 3-D.It is important to know the wealth of information concern-ing cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g.,
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Surgery_Schwartz. culture has become one of the most power-ful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and cellular biology.32 Through their ability to be maintained in vitro, cells can be manipulated by the introduc-tion of genes of interest (cell transfection) and be transferred into in vivo biologic receivers (cell transplantation) to study the biologic effect of the interested genes (Fig. 15-21). In common Brunicardi_Ch15_p0479-p0510.indd 49918/02/19 11:12 AM 500BASIC CONSIDERATIONSPART Ilaboratory settings, cells are cultured either as a monolayer (in which cells grow as one layer on culture dishes), considered 2-D, or in suspension or biomedical material skeleton such as hydrogel, considered 3-D.It is important to know the wealth of information concern-ing cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g.,
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Surgery_Schwartz_3406
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Surgery_Schwartz
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is important to know the wealth of information concern-ing cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g., origins of the cells such as epithelial or fibroblasts, or pri-mary vs immortalized/transformed cells). It is also necessary to use a cell type-specific culture medium that varies in combi-nation of growth factors and serum concentrations. If primary cells are derived from human patients or animals, some com-mercial resources have a variety of culture media available for testing. Generally, cells are manipulated in a sterile hood, and the working surfaces are wiped with 70% to 80% ethyl alcohol solution. Cultured cells are usually maintained in a humidified 5% carbon dioxide incubator at 37°C (98.6°F) to maintain a Ph value raging from 7.2 ∼ 7.4 and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). In
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Surgery_Schwartz. is important to know the wealth of information concern-ing cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g., origins of the cells such as epithelial or fibroblasts, or pri-mary vs immortalized/transformed cells). It is also necessary to use a cell type-specific culture medium that varies in combi-nation of growth factors and serum concentrations. If primary cells are derived from human patients or animals, some com-mercial resources have a variety of culture media available for testing. Generally, cells are manipulated in a sterile hood, and the working surfaces are wiped with 70% to 80% ethyl alcohol solution. Cultured cells are usually maintained in a humidified 5% carbon dioxide incubator at 37°C (98.6°F) to maintain a Ph value raging from 7.2 ∼ 7.4 and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). In
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Surgery_Schwartz_3407
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Surgery_Schwartz
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to maintain a Ph value raging from 7.2 ∼ 7.4 and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). In some cases, cells need to be maintained in hypoxia, and the oxygen input could be reduced to as low as 1%. As a general rule, cells should be fed with fresh medium every 2 to 3 days and split when they reach confluency. Depending on the growth rate of cells, the actual time and number of plates required to split cells in two varies from cell line to cell line. Splitting a monolayer requires the detachment of cells from plates by using a trypsin or colla-genase treatment, of which concentration and time period vary depending on cell lines. If cultured cells grow continuously in suspension, they are split or subcultured by dilution.Because cell lines may change their properties when cul-tured, it is not possible to maintain cell lines in culture indefi-nitely. Therefore, it is essential to store
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Surgery_Schwartz. to maintain a Ph value raging from 7.2 ∼ 7.4 and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). In some cases, cells need to be maintained in hypoxia, and the oxygen input could be reduced to as low as 1%. As a general rule, cells should be fed with fresh medium every 2 to 3 days and split when they reach confluency. Depending on the growth rate of cells, the actual time and number of plates required to split cells in two varies from cell line to cell line. Splitting a monolayer requires the detachment of cells from plates by using a trypsin or colla-genase treatment, of which concentration and time period vary depending on cell lines. If cultured cells grow continuously in suspension, they are split or subcultured by dilution.Because cell lines may change their properties when cul-tured, it is not possible to maintain cell lines in culture indefi-nitely. Therefore, it is essential to store
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Surgery_Schwartz_3408
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Surgery_Schwartz
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split or subcultured by dilution.Because cell lines may change their properties when cul-tured, it is not possible to maintain cell lines in culture indefi-nitely. Therefore, it is essential to store cells at various time passages for future use. The common procedure to use is cryo-preservation. The solution for cryopreservation is usually fetal calf serum containing 10% dimethyl sulfoxide or glycerol, stored in liquid nitrogen (−196°C [−320.8°F]) for years of pres-ervation. However, the viability and health of cells when thawed will decrease over time even in liquid nitrogen.Cell Transfection. Cells are cultured for two reasons: to main-tain and to manipulate them (see Fig. 15-21). The transfer of foreign macromolecules, such as nucleic acid, into living cells provides an efficient method for studying a variety of cellular processes and functions at the molecular level. DNA transfec-tion has become an important tool for studying the regulation and function of genes. The cDNA to be
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Surgery_Schwartz. split or subcultured by dilution.Because cell lines may change their properties when cul-tured, it is not possible to maintain cell lines in culture indefi-nitely. Therefore, it is essential to store cells at various time passages for future use. The common procedure to use is cryo-preservation. The solution for cryopreservation is usually fetal calf serum containing 10% dimethyl sulfoxide or glycerol, stored in liquid nitrogen (−196°C [−320.8°F]) for years of pres-ervation. However, the viability and health of cells when thawed will decrease over time even in liquid nitrogen.Cell Transfection. Cells are cultured for two reasons: to main-tain and to manipulate them (see Fig. 15-21). The transfer of foreign macromolecules, such as nucleic acid, into living cells provides an efficient method for studying a variety of cellular processes and functions at the molecular level. DNA transfec-tion has become an important tool for studying the regulation and function of genes. The cDNA to be
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Surgery_Schwartz_3409
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Surgery_Schwartz
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for studying a variety of cellular processes and functions at the molecular level. DNA transfec-tion has become an important tool for studying the regulation and function of genes. The cDNA to be expressed should be in a plasmid vector, behind an appropriate promoter working in mammalian cells (e.g., the constitutively active cytomegalo-virus promoter or inducible promoter). Depending on the cell type, many ways of introducing DNA into mammalian cells have been developed. Commonly used approaches include cal-cium phosphate, electroporation, liposome-mediated transfec-tion, the nonliposomal formulation, and the use of viral vectors. These methods have shown variable success when attempting to transfect a wide variety of cells. Transfection can be performed in the presence or absence of serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to
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Surgery_Schwartz. for studying a variety of cellular processes and functions at the molecular level. DNA transfec-tion has become an important tool for studying the regulation and function of genes. The cDNA to be expressed should be in a plasmid vector, behind an appropriate promoter working in mammalian cells (e.g., the constitutively active cytomegalo-virus promoter or inducible promoter). Depending on the cell type, many ways of introducing DNA into mammalian cells have been developed. Commonly used approaches include cal-cium phosphate, electroporation, liposome-mediated transfec-tion, the nonliposomal formulation, and the use of viral vectors. These methods have shown variable success when attempting to transfect a wide variety of cells. Transfection can be performed in the presence or absence of serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to
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Surgery_Schwartz_3410
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Surgery_Schwartz
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serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to follow the manufacturer’s instructions for the particular reagent. General considerations for a successful transfection depend on several parameters, such as the quality and quantity of DNA and cell culture (type of cell and growth phase). To minimize variations in both of these in transfection experiments, it is best to use cells that are healthy, proliferate well, and are plated at a constant density.Depending on the transfection method, DNA expression can be transient or stable. Using calcium phosphate and lipo-some-mediated transfection, after DNA is introduced into the cells it is normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, func-tional assays should be performed 24 to 72 hours after transfec-tion, also termed
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Surgery_Schwartz. serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to follow the manufacturer’s instructions for the particular reagent. General considerations for a successful transfection depend on several parameters, such as the quality and quantity of DNA and cell culture (type of cell and growth phase). To minimize variations in both of these in transfection experiments, it is best to use cells that are healthy, proliferate well, and are plated at a constant density.Depending on the transfection method, DNA expression can be transient or stable. Using calcium phosphate and lipo-some-mediated transfection, after DNA is introduced into the cells it is normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, func-tional assays should be performed 24 to 72 hours after transfec-tion, also termed
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Surgery_Schwartz_3411
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Surgery_Schwartz
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normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, func-tional assays should be performed 24 to 72 hours after transfec-tion, also termed transient transfection. In many applications, it is important to study the long-term effects of DNA in cells by stable transfection. Thus, electroporation and viral vector are often used in these situations to enable integration of ectopic DNA into the host genome. Stable cell clones can be selected when plasmids carry an antibiotic-resistant marker. In the pres-ence of antibiotics, only those cells that continuously carry the antibiotic-resistant marker (after generations of cell division) can survive. One application of stable transfection is the gen-eration of transgenic or knockout mouse models, in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells
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Surgery_Schwartz. normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, func-tional assays should be performed 24 to 72 hours after transfec-tion, also termed transient transfection. In many applications, it is important to study the long-term effects of DNA in cells by stable transfection. Thus, electroporation and viral vector are often used in these situations to enable integration of ectopic DNA into the host genome. Stable cell clones can be selected when plasmids carry an antibiotic-resistant marker. In the pres-ence of antibiotics, only those cells that continuously carry the antibiotic-resistant marker (after generations of cell division) can survive. One application of stable transfection is the gen-eration of transgenic or knockout mouse models, in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells
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Surgery_Schwartz_3412
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Surgery_Schwartz
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in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells also can be transplanted into host organs to test the effect of transgenic cells in vivo.Genetic ManipulationsUnderstanding how genes control the growth and differentia-tion of the mammalian organism has been the most challenging topic of modern research. It is essential for us to understand how genetic mutations and chemicals lead to the pathologic condi-tion of human bodies. The knowledge and ability to change the genetic program will inevitably make a great impact on society and have far-reaching effects on how we think of ourselves.The mouse has become firmly established as the primary experimental model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.33 The gene
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Surgery_Schwartz. in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells also can be transplanted into host organs to test the effect of transgenic cells in vivo.Genetic ManipulationsUnderstanding how genes control the growth and differentia-tion of the mammalian organism has been the most challenging topic of modern research. It is essential for us to understand how genetic mutations and chemicals lead to the pathologic condi-tion of human bodies. The knowledge and ability to change the genetic program will inevitably make a great impact on society and have far-reaching effects on how we think of ourselves.The mouse has become firmly established as the primary experimental model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.33 The gene
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Surgery_Schwartz_3413
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Surgery_Schwartz
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model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.33 The gene function can be studied by cre-ating mutant mice through homologous recombination (gene knockout). A gene of interest (GOI) also can be intro-duced into the mouse (transgenic mouse) to study its effect on development or diseases. Because mouse models do not pre-cisely represent human biology, genetic manipulations of human somatic or ES cells provide a great means for the understanding of the molecular networks in human cells in addition to mouse models. In all cases, the gene to be manipulated must first be cloned. Gene cloning has been made easy by recombinant DNA technology and the availability of human and mouse genomes (see “Human Genome”). The following section briefly describes the technologies and the principles behind combining both mouse genetics and human cell culture to explore
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Surgery_Schwartz. model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.33 The gene function can be studied by cre-ating mutant mice through homologous recombination (gene knockout). A gene of interest (GOI) also can be intro-duced into the mouse (transgenic mouse) to study its effect on development or diseases. Because mouse models do not pre-cisely represent human biology, genetic manipulations of human somatic or ES cells provide a great means for the understanding of the molecular networks in human cells in addition to mouse models. In all cases, the gene to be manipulated must first be cloned. Gene cloning has been made easy by recombinant DNA technology and the availability of human and mouse genomes (see “Human Genome”). The following section briefly describes the technologies and the principles behind combining both mouse genetics and human cell culture to explore
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Surgery_Schwartz_3414
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Surgery_Schwartz
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of human and mouse genomes (see “Human Genome”). The following section briefly describes the technologies and the principles behind combining both mouse genetics and human cell culture to explore gene function and disease mechanisms.Transgenic Mice. During the past 20 years, DNA cloning and other techniques have allowed the introduction of new genetic material into the mouse germline. As early as 1980, the first genetic material was successfully introduced into the mouse germline by using pronuclear microinjection of DNA (Fig. 15-22). These animals, called transgenic, contain for-eign DNA within their genomes. In simple terms, a transgenic mouse is created by the microinjection of ectopic DNA into the one-celled mouse embryo to induce integration, allowing the 4Brunicardi_Ch15_p0479-p0510.indd 50018/02/19 11:12 AM 501MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15DNAPronucleusDNA microinjected intopronucleus of fertilized eggFoster
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Surgery_Schwartz. of human and mouse genomes (see “Human Genome”). The following section briefly describes the technologies and the principles behind combining both mouse genetics and human cell culture to explore gene function and disease mechanisms.Transgenic Mice. During the past 20 years, DNA cloning and other techniques have allowed the introduction of new genetic material into the mouse germline. As early as 1980, the first genetic material was successfully introduced into the mouse germline by using pronuclear microinjection of DNA (Fig. 15-22). These animals, called transgenic, contain for-eign DNA within their genomes. In simple terms, a transgenic mouse is created by the microinjection of ectopic DNA into the one-celled mouse embryo to induce integration, allowing the 4Brunicardi_Ch15_p0479-p0510.indd 50018/02/19 11:12 AM 501MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15DNAPronucleusDNA microinjected intopronucleus of fertilized eggFoster
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50018/02/19 11:12 AM 501MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15DNAPronucleusDNA microinjected intopronucleus of fertilized eggFoster mothercarryingmicroinjected DNATransgenicmouseFigure 15-22. Transgenic mouse tech-nology. DNA is microinjected into a pro-nucleus of a fertilized egg, which is then transplanted into a foster mother. The microinjected egg develops offspring mice. Incorporation of the injected DNA into offspring is indicated by the differ-ent coat color of offspring mice.efficient introduction of cloned genes into the following devel-oping mouse somatic tissues, as well as into the germline.Designs of a Transgene The transgenic technique has proven to be extremely important for basic investigations of gene regu-lation, creation of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct
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Surgery_Schwartz. 50018/02/19 11:12 AM 501MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15DNAPronucleusDNA microinjected intopronucleus of fertilized eggFoster mothercarryingmicroinjected DNATransgenicmouseFigure 15-22. Transgenic mouse tech-nology. DNA is microinjected into a pro-nucleus of a fertilized egg, which is then transplanted into a foster mother. The microinjected egg develops offspring mice. Incorporation of the injected DNA into offspring is indicated by the differ-ent coat color of offspring mice.efficient introduction of cloned genes into the following devel-oping mouse somatic tissues, as well as into the germline.Designs of a Transgene The transgenic technique has proven to be extremely important for basic investigations of gene regu-lation, creation of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct
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Surgery_Schwartz_3416
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Surgery_Schwartz
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of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct consists of a protein-encoding gene and a promoter that precedes it. The most common applications for the use of transgenic mice are similar to those in the cell culture system: (a) to study the functions of proteins encoded by the transgene, (b) to analyze the tissue-specific and developmental-stage–specific activity of a gene promoter, and (c) to generate reporter lines to facilitate biomedical studies. Examples of the first application include overexpression of oncogenes, growth factors, hormones, and other key regulatory genes, as well as genes of viral origins. Overexpression of the transgene normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate
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Surgery_Schwartz. of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct consists of a protein-encoding gene and a promoter that precedes it. The most common applications for the use of transgenic mice are similar to those in the cell culture system: (a) to study the functions of proteins encoded by the transgene, (b) to analyze the tissue-specific and developmental-stage–specific activity of a gene promoter, and (c) to generate reporter lines to facilitate biomedical studies. Examples of the first application include overexpression of oncogenes, growth factors, hormones, and other key regulatory genes, as well as genes of viral origins. Overexpression of the transgene normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate
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Surgery_Schwartz_3417
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normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate vicin-ity of the genes themselves. Thus, controlled expression of the transgene can be made possible by using an inducible or tissue-specific promoter. Furthermore, transgenic mice carry-ing dominant negative mutations of a regulatory gene also have been generated. For example, a truncated growth factor receptor that can bind to the ligand, but loses its catalytic activity when expressed in mice, can block the growth factor binding to the endogenous protein. In this way, the transgenic mice exhibit a loss of function of phenotype, possibly resembling the knockout of the endogenous gene. The second application of the trans-genic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called
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Surgery_Schwartz. normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate vicin-ity of the genes themselves. Thus, controlled expression of the transgene can be made possible by using an inducible or tissue-specific promoter. Furthermore, transgenic mice carry-ing dominant negative mutations of a regulatory gene also have been generated. For example, a truncated growth factor receptor that can bind to the ligand, but loses its catalytic activity when expressed in mice, can block the growth factor binding to the endogenous protein. In this way, the transgenic mice exhibit a loss of function of phenotype, possibly resembling the knockout of the endogenous gene. The second application of the trans-genic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called
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Surgery_Schwartz_3418
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application of the trans-genic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called LacZ), luciferase, or green fluorescence protein. Chemical staining of LacZ activity or detection of chemiluminescence/fluorescence can easily visu-alize the expression of the reporter gene. The third application originates from the second: when the activity of the promoter is known, a fluorescent reporter gene (such as GFP) will be driven by the tissue-specific promoter, therefore labeling a particular type of cells at a particular stage. This application is generally used to isolate a special cell type expressing the GFP reporter by fluorescence-activated cell sorting (FACS), as well as lineage-tracing experiments.Production of Transgenic Mice The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For
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Surgery_Schwartz. application of the trans-genic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called LacZ), luciferase, or green fluorescence protein. Chemical staining of LacZ activity or detection of chemiluminescence/fluorescence can easily visu-alize the expression of the reporter gene. The third application originates from the second: when the activity of the promoter is known, a fluorescent reporter gene (such as GFP) will be driven by the tissue-specific promoter, therefore labeling a particular type of cells at a particular stage. This application is generally used to isolate a special cell type expressing the GFP reporter by fluorescence-activated cell sorting (FACS), as well as lineage-tracing experiments.Production of Transgenic Mice The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For
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Surgery_Schwartz_3419
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experiments.Production of Transgenic Mice The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For DNA to be microinjected into mouse embryos, it should be linear-ized by restriction digestion to increase the chance of proper transgene integration. Concentration of DNA should be accu-rately determined. Mice that develop from injected eggs often are termed founder mice.Genotyping of Transgenic Mice The screening of founder mice and the transgenic lines derived from the founders is accomplished by determining the integration of the injected gene into the genome. This normally is achieved by performing PCR or Southern blot analysis with a small amount of DNA extracted from the mouse tail. Once a given founder mouse is identified to be transgenic, it will be mated to begin establish-ing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the
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Surgery_Schwartz. experiments.Production of Transgenic Mice The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For DNA to be microinjected into mouse embryos, it should be linear-ized by restriction digestion to increase the chance of proper transgene integration. Concentration of DNA should be accu-rately determined. Mice that develop from injected eggs often are termed founder mice.Genotyping of Transgenic Mice The screening of founder mice and the transgenic lines derived from the founders is accomplished by determining the integration of the injected gene into the genome. This normally is achieved by performing PCR or Southern blot analysis with a small amount of DNA extracted from the mouse tail. Once a given founder mouse is identified to be transgenic, it will be mated to begin establish-ing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the
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Surgery_Schwartz_3420
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given founder mouse is identified to be transgenic, it will be mated to begin establish-ing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the phenotype is due to transgene but not to the interruption of the gene where the transgene integrates into.Analysis of Phenotype of Transgenic Mice Phenotypes of transgenic mice are dictated by both the expression pattern and biologic functions of the transgene. Depending on the promoter and the transgene, phenotypes can be predictable or unpredict-able. Elucidation of the functions of the transgene-encoded pro-tein in vitro often offers some clue to what the protein might function to do in vivo. When a constitutively active promoter is used to drive the expression of transgenes, mice should express the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in dis-ease pathogenesis. Ideally, the use of tissue-specific or induc-ible
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Surgery_Schwartz. given founder mouse is identified to be transgenic, it will be mated to begin establish-ing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the phenotype is due to transgene but not to the interruption of the gene where the transgene integrates into.Analysis of Phenotype of Transgenic Mice Phenotypes of transgenic mice are dictated by both the expression pattern and biologic functions of the transgene. Depending on the promoter and the transgene, phenotypes can be predictable or unpredict-able. Elucidation of the functions of the transgene-encoded pro-tein in vitro often offers some clue to what the protein might function to do in vivo. When a constitutively active promoter is used to drive the expression of transgenes, mice should express the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in dis-ease pathogenesis. Ideally, the use of tissue-specific or induc-ible
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the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in dis-ease pathogenesis. Ideally, the use of tissue-specific or induc-ible promoter allows one to determine if the pathogenic protein leads to a reversible or irreversible disease process in a cell-autonomous manner. For example, rat insulin promoter can tar-get transgene expression exclusively in the β-cells of pancreatic islets. The phenotype of insulin promoter-mediated transgenic mice is projected to affect the function of human β-cells.Gene Knockout in Mice. The first recorded knockout mouse was created by Mario R. Capecchi, Sir Martin J. Evans, and Oliver Smithies in 1989. They were awarded the 2007 Nobel Prize in Physiology or Medicine. The isolation and genetic manipulation of mouse ES cells represents one of the most important milestones for modern genetic technologies.34 Sev-eral unique properties of ES cells, such as the pluripotency to differentiate into
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Surgery_Schwartz. the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in dis-ease pathogenesis. Ideally, the use of tissue-specific or induc-ible promoter allows one to determine if the pathogenic protein leads to a reversible or irreversible disease process in a cell-autonomous manner. For example, rat insulin promoter can tar-get transgene expression exclusively in the β-cells of pancreatic islets. The phenotype of insulin promoter-mediated transgenic mice is projected to affect the function of human β-cells.Gene Knockout in Mice. The first recorded knockout mouse was created by Mario R. Capecchi, Sir Martin J. Evans, and Oliver Smithies in 1989. They were awarded the 2007 Nobel Prize in Physiology or Medicine. The isolation and genetic manipulation of mouse ES cells represents one of the most important milestones for modern genetic technologies.34 Sev-eral unique properties of ES cells, such as the pluripotency to differentiate into
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of mouse ES cells represents one of the most important milestones for modern genetic technologies.34 Sev-eral unique properties of ES cells, such as the pluripotency to differentiate into all germ layers in an embryo, including the germline, make them an efficient vehicle to introduce genetic alterations in mice. An important breakthrough from this idea is to generate gene-targeted mutation in mice, first by introduc-ing the targeting vector into the ES cells, allowing selection Brunicardi_Ch15_p0479-p0510.indd 50118/02/19 11:12 AM 502BASIC CONSIDERATIONSPART IES cells growingin tissue cultureAltered versionof target geneconstructed bygenetic engineeringLet each cellgrow to forma colonyTest for the rarecolony in whichthe DNA fragmenthas replaced one copy of the normal geneES cells with one copy of target genereplaced by mutant geneInjectES cellsintoearly embryoFemale mouseMate and wait3 daysIsolatedearlyembryoEarly embryo partlyformed fromES cellsIntroduceearly embryo
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Surgery_Schwartz. of mouse ES cells represents one of the most important milestones for modern genetic technologies.34 Sev-eral unique properties of ES cells, such as the pluripotency to differentiate into all germ layers in an embryo, including the germline, make them an efficient vehicle to introduce genetic alterations in mice. An important breakthrough from this idea is to generate gene-targeted mutation in mice, first by introduc-ing the targeting vector into the ES cells, allowing selection Brunicardi_Ch15_p0479-p0510.indd 50118/02/19 11:12 AM 502BASIC CONSIDERATIONSPART IES cells growingin tissue cultureAltered versionof target geneconstructed bygenetic engineeringLet each cellgrow to forma colonyTest for the rarecolony in whichthe DNA fragmenthas replaced one copy of the normal geneES cells with one copy of target genereplaced by mutant geneInjectES cellsintoearly embryoFemale mouseMate and wait3 daysIsolatedearlyembryoEarly embryo partlyformed fromES cellsIntroduceearly embryo
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cells with one copy of target genereplaced by mutant geneInjectES cellsintoearly embryoFemale mouseMate and wait3 daysIsolatedearlyembryoEarly embryo partlyformed fromES cellsIntroduceearly embryo intopseudopregnantmouseSomatic cellsof offspring tested forpresence ofaltered gene, and selected mice bredto test for genein germline cellsTransgenic mouse with one copy of target genereplaced by altered gene in germlineABBirthIntroduce a DNA fragmentcontaining altered geneinto many cellsFigure 15-23. Knockout mouse technology. Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured embryonic stem (ES) cells. Only a few rare ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered
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Surgery_Schwartz. cells with one copy of target genereplaced by mutant geneInjectES cellsintoearly embryoFemale mouseMate and wait3 daysIsolatedearlyembryoEarly embryo partlyformed fromES cellsIntroduceearly embryo intopseudopregnantmouseSomatic cellsof offspring tested forpresence ofaltered gene, and selected mice bredto test for genein germline cellsTransgenic mouse with one copy of target genereplaced by altered gene in germlineABBirthIntroduce a DNA fragmentcontaining altered geneinto many cellsFigure 15-23. Knockout mouse technology. Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured embryonic stem (ES) cells. Only a few rare ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered
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gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells that carry the altered gene. Some of these mice also will contain germline cells that contain the altered gene. When bred with a normal mouse, some of the progeny of these mice will contain the altered gene in all of their cells. If two such mice are in turn bred (not shown), some of the progeny will contain two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such
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Surgery_Schwartz. gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells that carry the altered gene. Some of these mice also will contain germline cells that contain the altered gene. When bred with a normal mouse, some of the progeny of these mice will contain the altered gene in all of their cells. If two such mice are in turn bred (not shown), some of the progeny will contain two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such
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two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such mice are missing genes that function during development, they often die with specific defects long before they reach adulthood. These defects are carefully analyzed to help decipher the normal function of the missing gene. (Reproduced with permission from Alberts B, Johnson A, Lewis J, et al: Molecular Biology of the Cell, 6th ed. New York, NY: WW Norton Company; 2015.)for successful homologous recombination in a dish, then intro-ducing the selected ES clone into the blastocysts, and finally recovering animals bearing the mutant allele from the germline (Fig. 15-23). This not only makes mouse genetics a powerful approach to address important gene functions, but also identifies the mouse as a great system to model human disease.Targeting Vector The basic concept in building a target vec-tor to
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Surgery_Schwartz. two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such mice are missing genes that function during development, they often die with specific defects long before they reach adulthood. These defects are carefully analyzed to help decipher the normal function of the missing gene. (Reproduced with permission from Alberts B, Johnson A, Lewis J, et al: Molecular Biology of the Cell, 6th ed. New York, NY: WW Norton Company; 2015.)for successful homologous recombination in a dish, then intro-ducing the selected ES clone into the blastocysts, and finally recovering animals bearing the mutant allele from the germline (Fig. 15-23). This not only makes mouse genetics a powerful approach to address important gene functions, but also identifies the mouse as a great system to model human disease.Targeting Vector The basic concept in building a target vec-tor to
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a powerful approach to address important gene functions, but also identifies the mouse as a great system to model human disease.Targeting Vector The basic concept in building a target vec-tor to knock out a gene is to use two segments of homologous sequence to a GOI that flank a part of the gene essential for functions (e.g., the coding region). In the targeting vector, a pos-itive selectable marker (e.g., the neo gene) is placed between the homology arms. Upon the homologous recombination between the arms of the vector and the corresponding genomic regions of the GOI in ES cells, the positive selectable marker will replace the essential segment of the target gene, thus creating a null allele. In addition, a negative selectable marker also can Brunicardi_Ch15_p0479-p0510.indd 50218/02/19 11:12 AM 503MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15be used alone or in combination with the positive selectable marker, but must be placed outside of the
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Surgery_Schwartz. a powerful approach to address important gene functions, but also identifies the mouse as a great system to model human disease.Targeting Vector The basic concept in building a target vec-tor to knock out a gene is to use two segments of homologous sequence to a GOI that flank a part of the gene essential for functions (e.g., the coding region). In the targeting vector, a pos-itive selectable marker (e.g., the neo gene) is placed between the homology arms. Upon the homologous recombination between the arms of the vector and the corresponding genomic regions of the GOI in ES cells, the positive selectable marker will replace the essential segment of the target gene, thus creating a null allele. In addition, a negative selectable marker also can Brunicardi_Ch15_p0479-p0510.indd 50218/02/19 11:12 AM 503MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15be used alone or in combination with the positive selectable marker, but must be placed outside of the
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11:12 AM 503MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15be used alone or in combination with the positive selectable marker, but must be placed outside of the homologous arms to enrich for homologous recombination. To create a conditional knockout (i.e., gene knockout in a spatiotemporal fashion), site-specific recombinases such as the popular cre-loxP system are used. If the consensus loxP sequences that are recognized by cre recombinases are properly designed into targeting loci, con-trolled expression of the recombinase as a transgene can result in the site-specific recombination at the right time and in the right place (i.e., cell type or tissue). This method, often referred to as conditional knockout, is markedly useful to prevent devel-opmental compensations and to introduce null mutations in the adult mouse that would otherwise be lethal. To bring in addi-tional control to tissue-specific cre, an inducible cre could be adopted on top of the
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Surgery_Schwartz. 11:12 AM 503MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15be used alone or in combination with the positive selectable marker, but must be placed outside of the homologous arms to enrich for homologous recombination. To create a conditional knockout (i.e., gene knockout in a spatiotemporal fashion), site-specific recombinases such as the popular cre-loxP system are used. If the consensus loxP sequences that are recognized by cre recombinases are properly designed into targeting loci, con-trolled expression of the recombinase as a transgene can result in the site-specific recombination at the right time and in the right place (i.e., cell type or tissue). This method, often referred to as conditional knockout, is markedly useful to prevent devel-opmental compensations and to introduce null mutations in the adult mouse that would otherwise be lethal. To bring in addi-tional control to tissue-specific cre, an inducible cre could be adopted on top of the
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and to introduce null mutations in the adult mouse that would otherwise be lethal. To bring in addi-tional control to tissue-specific cre, an inducible cre could be adopted on top of the tissue-specific promoter. The most popu-lar inducible cre includes CreER: CreER encodes a cre fused with estrogen receptor located to cytosol. With the signaling of tamoxifen, CreER is released and then translocates into the nucleus to induce the recombination of loxP. Therefore, the tim-ing of recombination could be precisely determined by control-ling the time of administering tamoxifen. Overall, this cre-loxP system allows for spatial and temporal control over transgene expression and takes advantage of inducers with minimal pleio-tropic effects.Introduction of the Targeting Vector into ES Cells ES cell lines can be obtained from other investigators or commer-cial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, opti-mal growth
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Surgery_Schwartz. and to introduce null mutations in the adult mouse that would otherwise be lethal. To bring in addi-tional control to tissue-specific cre, an inducible cre could be adopted on top of the tissue-specific promoter. The most popu-lar inducible cre includes CreER: CreER encodes a cre fused with estrogen receptor located to cytosol. With the signaling of tamoxifen, CreER is released and then translocates into the nucleus to induce the recombination of loxP. Therefore, the tim-ing of recombination could be precisely determined by control-ling the time of administering tamoxifen. Overall, this cre-loxP system allows for spatial and temporal control over transgene expression and takes advantage of inducers with minimal pleio-tropic effects.Introduction of the Targeting Vector into ES Cells ES cell lines can be obtained from other investigators or commer-cial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, opti-mal growth
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cell lines can be obtained from other investigators or commer-cial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, opti-mal growth conditions should be provided in culture. If cul-ture conditions are inappropriate or inadequate, ES cells may acquire genetic lesions or alter their gene expression patterns and consequently decrease their pluripotency. Excellent proto-cols are available in public domains or in mouse facilities in most institutions.To alter the genome of ES cells, the targeting vector DNA then is transfected into ES cells. Electroporation is the most widely used and the most efficient transfection method for ES cells. Similar procedures for stable cell transfection are used for selecting ES cells that carry the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a
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Surgery_Schwartz. cell lines can be obtained from other investigators or commer-cial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, opti-mal growth conditions should be provided in culture. If cul-ture conditions are inappropriate or inadequate, ES cells may acquire genetic lesions or alter their gene expression patterns and consequently decrease their pluripotency. Excellent proto-cols are available in public domains or in mouse facilities in most institutions.To alter the genome of ES cells, the targeting vector DNA then is transfected into ES cells. Electroporation is the most widely used and the most efficient transfection method for ES cells. Similar procedures for stable cell transfection are used for selecting ES cells that carry the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a
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the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a positive selectable antibiotic drug. After a certain period of time and depending on the type of antibiotics, all sensitive cells die, and the resistant cells grow into individual colonies of the appropriate size for subcloning by picking. It is extremely important to minimize the time dur-ing which ES cells are in culture between selection and injection into blastocysts. Before injecting the ES cells, DNA is prepared from ES colonies to screen for positive ES cells that exhibit the correct integration or homologous recombination of the target-ing vector. Positive ES colonies are then expanded and used for creation of chimeras.Creation of the Chimera A chimeric organism is one in which cells originate from more than one embryo source. Here, chi-meric mice are denoted as those that contain
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Surgery_Schwartz. the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a positive selectable antibiotic drug. After a certain period of time and depending on the type of antibiotics, all sensitive cells die, and the resistant cells grow into individual colonies of the appropriate size for subcloning by picking. It is extremely important to minimize the time dur-ing which ES cells are in culture between selection and injection into blastocysts. Before injecting the ES cells, DNA is prepared from ES colonies to screen for positive ES cells that exhibit the correct integration or homologous recombination of the target-ing vector. Positive ES colonies are then expanded and used for creation of chimeras.Creation of the Chimera A chimeric organism is one in which cells originate from more than one embryo source. Here, chi-meric mice are denoted as those that contain
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and used for creation of chimeras.Creation of the Chimera A chimeric organism is one in which cells originate from more than one embryo source. Here, chi-meric mice are denoted as those that contain some tissues from the ES cells with an altered genome. When these ES cells give rise to the lineage of the germ layer, the germ cells carrying the altered genome can be passed on to the offspring, thus creating the germline transmission from ES cells. There are two methods for introducing ES cells into preimplantation-stage embryos: injection and aggregation. The injection of embryonic cells directly into the cavity of blastocysts is one of the fundamen-tal methods for generating chimeras, but aggregation chimeras also have become an important alternative for transmitting the ES cell genome into mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of rec-ognizable markers (e.g., coat color) that are specific to the donor mouse and the ES cells
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Surgery_Schwartz. and used for creation of chimeras.Creation of the Chimera A chimeric organism is one in which cells originate from more than one embryo source. Here, chi-meric mice are denoted as those that contain some tissues from the ES cells with an altered genome. When these ES cells give rise to the lineage of the germ layer, the germ cells carrying the altered genome can be passed on to the offspring, thus creating the germline transmission from ES cells. There are two methods for introducing ES cells into preimplantation-stage embryos: injection and aggregation. The injection of embryonic cells directly into the cavity of blastocysts is one of the fundamen-tal methods for generating chimeras, but aggregation chimeras also have become an important alternative for transmitting the ES cell genome into mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of rec-ognizable markers (e.g., coat color) that are specific to the donor mouse and the ES cells
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mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of rec-ognizable markers (e.g., coat color) that are specific to the donor mouse and the ES cells can be used to identify chimeric mice. However, most experimenters probably use existing mouse core facilities already established in some institutions or contract a commercial vendor for the creation of a chimera.Genotyping and Phenotyping of Knockout Animals The next step is to analyze whether germline transmission of tar-geted mutation occurs in mice. DNA from a small amount of tissue from offspring of the chimera is extracted and subjected to genomic PCR or Southern blot DNA hybridization. Positive mice (i.e., those with properly integrated targeting vector into the genome) will be used for the propagation of more knockout mice for phenotype analysis. When the knockout genes are cru-cial for early embryogenesis, mice often die in utero, an occur-rence called embryonic lethality. When
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Surgery_Schwartz. mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of rec-ognizable markers (e.g., coat color) that are specific to the donor mouse and the ES cells can be used to identify chimeric mice. However, most experimenters probably use existing mouse core facilities already established in some institutions or contract a commercial vendor for the creation of a chimera.Genotyping and Phenotyping of Knockout Animals The next step is to analyze whether germline transmission of tar-geted mutation occurs in mice. DNA from a small amount of tissue from offspring of the chimera is extracted and subjected to genomic PCR or Southern blot DNA hybridization. Positive mice (i.e., those with properly integrated targeting vector into the genome) will be used for the propagation of more knockout mice for phenotype analysis. When the knockout genes are cru-cial for early embryogenesis, mice often die in utero, an occur-rence called embryonic lethality. When
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the propagation of more knockout mice for phenotype analysis. When the knockout genes are cru-cial for early embryogenesis, mice often die in utero, an occur-rence called embryonic lethality. When this happens, only the phenotype of the homozygous (both alleles ablated) knockout mouse embryos and the phenotype of the heterozygous (only one allele ablated) adult mice can be studied. Because most researchers are interested in the phenotype of adult mice, in par-ticular when using mice as disease models, it is recommended to create the conditional knockout using the cre-loxP system so that the GOI can be knocked out at will.To date, more than 5000 genes have been disrupted by homologous recombination and transmitted through the germline. The phenotypic studies of these mice provide ample information about the functions of these genes in growth and differentiation of organisms and during development of human diseases.RNA Interference. Although gene ablation in animal models provides an
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Surgery_Schwartz. the propagation of more knockout mice for phenotype analysis. When the knockout genes are cru-cial for early embryogenesis, mice often die in utero, an occur-rence called embryonic lethality. When this happens, only the phenotype of the homozygous (both alleles ablated) knockout mouse embryos and the phenotype of the heterozygous (only one allele ablated) adult mice can be studied. Because most researchers are interested in the phenotype of adult mice, in par-ticular when using mice as disease models, it is recommended to create the conditional knockout using the cre-loxP system so that the GOI can be knocked out at will.To date, more than 5000 genes have been disrupted by homologous recombination and transmitted through the germline. The phenotypic studies of these mice provide ample information about the functions of these genes in growth and differentiation of organisms and during development of human diseases.RNA Interference. Although gene ablation in animal models provides an
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about the functions of these genes in growth and differentiation of organisms and during development of human diseases.RNA Interference. Although gene ablation in animal models provides an important means to understand the in vivo functions of GOI, animal models may not adequately represent human biology. Alternatively, gene targeting can be used to knock out genes in human cells, including human ES cells. Gene targeting in human ES cells by homologous recombination has extremely low efficiency, although there are more new techniques emerg-ing aimed at increasing the targeting efficiency. A number of recent advances have made gene targeting in somatic cells as easy as in murine ES cells.33 However, gene targeting (knocking out both alleles) in somatic cells is a time-consuming process.Development of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.35 RNAi is an ancient natural mechanism
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Surgery_Schwartz. about the functions of these genes in growth and differentiation of organisms and during development of human diseases.RNA Interference. Although gene ablation in animal models provides an important means to understand the in vivo functions of GOI, animal models may not adequately represent human biology. Alternatively, gene targeting can be used to knock out genes in human cells, including human ES cells. Gene targeting in human ES cells by homologous recombination has extremely low efficiency, although there are more new techniques emerg-ing aimed at increasing the targeting efficiency. A number of recent advances have made gene targeting in somatic cells as easy as in murine ES cells.33 However, gene targeting (knocking out both alleles) in somatic cells is a time-consuming process.Development of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.35 RNAi is an ancient natural mechanism
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of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.35 RNAi is an ancient natural mechanism by which small, double-stranded RNA (dsRNA) acts as a guide for an enzyme complex that destroys complementary RNA and downregulates gene expres-sion in a sequence-specific manner. Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. In nonmammalian systems such as Drosophila, it appears that longer dsRNA is processed into 21–23 nt dsRNA (called small interfering RNA or siRNA) by an enzyme called Dicer contain-ing RNase III motifs. The siRNA apparently then acts as a guide sequence within a multicomponent nuclease complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in
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Surgery_Schwartz. of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.35 RNAi is an ancient natural mechanism by which small, double-stranded RNA (dsRNA) acts as a guide for an enzyme complex that destroys complementary RNA and downregulates gene expres-sion in a sequence-specific manner. Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. In nonmammalian systems such as Drosophila, it appears that longer dsRNA is processed into 21–23 nt dsRNA (called small interfering RNA or siRNA) by an enzyme called Dicer contain-ing RNase III motifs. The siRNA apparently then acts as a guide sequence within a multicomponent nuclease complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in
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complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in mammalian cells (Fig. 15-24).Brunicardi_Ch15_p0479-p0510.indd 50318/02/19 11:12 AM 504BASIC CONSIDERATIONSPART IPol IIITTTTTshRNAsiRNADicerRISCUUUUUUmRNAm7GAAAAAAmRNA targeted by siRNAUUm7GAAAAAAmRNA cleaved and degradedm7GAAAAAAFigure 15-24. RNA interference in mammalian cells. Small interfering RNA (siRNA) can be produced from a polymerase III–driven expres-sion vector. Such a vector first synthesizes a 19–29 nt double-stranded (ds)RNA stem and a loop (labeled as shRNA in the figure), and then the RNase complex called Dicer processes the hairpin RNA into a small dsRNA (labeled as siRNA in the figure). siRNA can be chemically synthesized and directly introduced into the target cell. In the cell, through RNA-induced silencing complex (RISC), siRNA recognizes and degrades target
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Surgery_Schwartz. complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in mammalian cells (Fig. 15-24).Brunicardi_Ch15_p0479-p0510.indd 50318/02/19 11:12 AM 504BASIC CONSIDERATIONSPART IPol IIITTTTTshRNAsiRNADicerRISCUUUUUUmRNAm7GAAAAAAmRNA targeted by siRNAUUm7GAAAAAAmRNA cleaved and degradedm7GAAAAAAFigure 15-24. RNA interference in mammalian cells. Small interfering RNA (siRNA) can be produced from a polymerase III–driven expres-sion vector. Such a vector first synthesizes a 19–29 nt double-stranded (ds)RNA stem and a loop (labeled as shRNA in the figure), and then the RNase complex called Dicer processes the hairpin RNA into a small dsRNA (labeled as siRNA in the figure). siRNA can be chemically synthesized and directly introduced into the target cell. In the cell, through RNA-induced silencing complex (RISC), siRNA recognizes and degrades target
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siRNA in the figure). siRNA can be chemically synthesized and directly introduced into the target cell. In the cell, through RNA-induced silencing complex (RISC), siRNA recognizes and degrades target messenger RNAs (mRNAs).For siRNA studies in mammalian cells, researchers have used two 21-mer RNAs with 19 complementary nucleotides and 3′ terminal noncomplementary dimers of thymidine or uri-dine. The antisense siRNA strand is fully complementary to the mRNA target sequence. Target sequences for an siRNA are identified visually or by software.The target 19 nucleotides should be compared to an appro-priate genome database to eliminate any sequences with sig-nificant homology to other genes. Those sequences that appear to be specific to the GOI are the potential siRNA target sites. A few of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA
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Surgery_Schwartz. siRNA in the figure). siRNA can be chemically synthesized and directly introduced into the target cell. In the cell, through RNA-induced silencing complex (RISC), siRNA recognizes and degrades target messenger RNAs (mRNAs).For siRNA studies in mammalian cells, researchers have used two 21-mer RNAs with 19 complementary nucleotides and 3′ terminal noncomplementary dimers of thymidine or uri-dine. The antisense siRNA strand is fully complementary to the mRNA target sequence. Target sequences for an siRNA are identified visually or by software.The target 19 nucleotides should be compared to an appro-priate genome database to eliminate any sequences with sig-nificant homology to other genes. Those sequences that appear to be specific to the GOI are the potential siRNA target sites. A few of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA
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of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA sequence. A deoxythymidine dimer is rou-tinely incorporated at the 3′ end of the sense strand siRNA, although it is unknown whether this noncomplementary dinu-cleotide is important for the activity of siRNAs.There are two ways to introduce siRNA to knock down gene expression in human cells:1. RNA transfection: siRNA can be made chemically or using an in vitro transcription method. Like DNA oligos, chemically synthesized siRNA oligos can be commercially ordered. However, synthetic siRNA is expensive, and sev-eral siRNAs may have to be tried before a particular gene is successfully silenced. In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as
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Surgery_Schwartz. of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA sequence. A deoxythymidine dimer is rou-tinely incorporated at the 3′ end of the sense strand siRNA, although it is unknown whether this noncomplementary dinu-cleotide is important for the activity of siRNAs.There are two ways to introduce siRNA to knock down gene expression in human cells:1. RNA transfection: siRNA can be made chemically or using an in vitro transcription method. Like DNA oligos, chemically synthesized siRNA oligos can be commercially ordered. However, synthetic siRNA is expensive, and sev-eral siRNAs may have to be tried before a particular gene is successfully silenced. In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as
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Surgery_Schwartz_3439
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Surgery_Schwartz
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In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as recom-binant Dicers will be used to process the long dsRNA into a mixture of 21–23 nt siRNA. siRNA oligos or mixtures can be transfected into a few characterized cell lines such as HeLa (human cervical carcinoma) and 293T cells (human kidney carcinoma). Transfection of siRNA directly into pri-mary cells may be difficult.2. DNA transfection: Expression vectors for expressing siRNA have been made using RNA polymerase III promoters such as U6 and H1. These promoters precisely transcribe a hairpin structure of dsRNA, which will be processed into siRNA in the cell (see Fig. 15-24). Therefore, properly designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advan-tages of the siRNA expression vectors over siRNA oligos. First, it is
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Surgery_Schwartz. In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as recom-binant Dicers will be used to process the long dsRNA into a mixture of 21–23 nt siRNA. siRNA oligos or mixtures can be transfected into a few characterized cell lines such as HeLa (human cervical carcinoma) and 293T cells (human kidney carcinoma). Transfection of siRNA directly into pri-mary cells may be difficult.2. DNA transfection: Expression vectors for expressing siRNA have been made using RNA polymerase III promoters such as U6 and H1. These promoters precisely transcribe a hairpin structure of dsRNA, which will be processed into siRNA in the cell (see Fig. 15-24). Therefore, properly designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advan-tages of the siRNA expression vectors over siRNA oligos. First, it is
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Surgery_Schwartz_3440
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designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advan-tages of the siRNA expression vectors over siRNA oligos. First, it is easier to transfect DNA into cells. Second, sta-ble populations of cells can be generated that maintain the long-term silencing of target genes. Furthermore, the siRNA expression cassette can be incorporated into a retroviral or adenoviral vector to provide a wide spectrum of applications in gene therapy.There has been a fast and fruitful development of RNAi tools for in vitro and in vivo use in mammals. These novel approaches, together with future developments, will be crucial to put RNAi technology to use for effective disease therapy or to exert the awesome power of mammalian genetics. Therefore, the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With
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Surgery_Schwartz. designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advan-tages of the siRNA expression vectors over siRNA oligos. First, it is easier to transfect DNA into cells. Second, sta-ble populations of cells can be generated that maintain the long-term silencing of target genes. Furthermore, the siRNA expression cassette can be incorporated into a retroviral or adenoviral vector to provide a wide spectrum of applications in gene therapy.There has been a fast and fruitful development of RNAi tools for in vitro and in vivo use in mammals. These novel approaches, together with future developments, will be crucial to put RNAi technology to use for effective disease therapy or to exert the awesome power of mammalian genetics. Therefore, the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With
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Surgery_Schwartz_3441
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the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With the availability of the human genome sequences, RNAi approaches hold tremendous promise for unleashing the dor-mant potential of sequenced genomes.Practical applications of RNAi will possibly result in new therapeutic interventions. In 2002, the concept of using siRNA in battling infectious diseases and carcinogenesis was proven effective. These include notable successes in blocking repli-cation of viruses, such as HIV, hepatitis B virus, and hepati-tis C virus, in cultured cells using siRNA targeted at the viral genome or the human gene encoding viral receptors. RNAi has been shown to antagonize the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential
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Surgery_Schwartz. the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With the availability of the human genome sequences, RNAi approaches hold tremendous promise for unleashing the dor-mant potential of sequenced genomes.Practical applications of RNAi will possibly result in new therapeutic interventions. In 2002, the concept of using siRNA in battling infectious diseases and carcinogenesis was proven effective. These include notable successes in blocking repli-cation of viruses, such as HIV, hepatitis B virus, and hepati-tis C virus, in cultured cells using siRNA targeted at the viral genome or the human gene encoding viral receptors. RNAi has been shown to antagonize the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential
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Surgery_Schwartz_3442
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the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential applications for some dominant genetic disorders.The 21st century, already heralded as the “century of the gene,” carries great promise for alleviating suffering from dis-ease and improving human health. On the whole, completion of Brunicardi_Ch15_p0479-p0510.indd 50418/02/19 11:12 AM 505MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15the human genome blueprint, the promise of gene therapy and molecular therapies, and the existence of stem cells have cap-tured the imagination of the public and the biomedical commu-nity. Aside from their potential in curing human diseases, these emerging technologies also have provoked many political, eco-nomic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention
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Surgery_Schwartz. the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential applications for some dominant genetic disorders.The 21st century, already heralded as the “century of the gene,” carries great promise for alleviating suffering from dis-ease and improving human health. On the whole, completion of Brunicardi_Ch15_p0479-p0510.indd 50418/02/19 11:12 AM 505MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15the human genome blueprint, the promise of gene therapy and molecular therapies, and the existence of stem cells have cap-tured the imagination of the public and the biomedical commu-nity. Aside from their potential in curing human diseases, these emerging technologies also have provoked many political, eco-nomic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention
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diseases, these emerging technologies also have provoked many political, eco-nomic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention must also be paid to concerns for their inherent risks and social impli-cations. It is important for surgeons to play a leadership role in the emergence of personalized medicine and surgery, as sur-geons have access to the diseased tissues. Surgeons should be establishing collaborations with the genomic and molecular sci-entists to develop genomic biobanks in order to study the genome and molecular signaling of the disease tissues that will help with an understanding of the underlying cause of an indi-vidual’s disease and ultimately lead to effective, targeted thera-pies. Surgeons must take this enormous opportunity to collaborate with basic and clinical scientists to develop the field of precision medicine and surgery this century.Bifunctional RNAi Technology.36 Over the last 20 years,
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Surgery_Schwartz. diseases, these emerging technologies also have provoked many political, eco-nomic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention must also be paid to concerns for their inherent risks and social impli-cations. It is important for surgeons to play a leadership role in the emergence of personalized medicine and surgery, as sur-geons have access to the diseased tissues. Surgeons should be establishing collaborations with the genomic and molecular sci-entists to develop genomic biobanks in order to study the genome and molecular signaling of the disease tissues that will help with an understanding of the underlying cause of an indi-vidual’s disease and ultimately lead to effective, targeted thera-pies. Surgeons must take this enormous opportunity to collaborate with basic and clinical scientists to develop the field of precision medicine and surgery this century.Bifunctional RNAi Technology.36 Over the last 20 years,
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this enormous opportunity to collaborate with basic and clinical scientists to develop the field of precision medicine and surgery this century.Bifunctional RNAi Technology.36 Over the last 20 years, the field has worked to define oncogene and nononcogene addic-tion, discriminate between driver and passenger genes, and appreciate the complexity of complex, robust, network interac-tions. These insights have led to a preliminary understanding of therapeutically relevant sensitivity and resistance pathway signal patterns requiring multiple target modulation. However, this knowledge has not been effectively or reproducibly clini-cally translated. Clinical response is usually far greater when a combination of single-target molecular therapy is administered. However, it must also be realized that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxic-ity has been demonstrated with combination
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Surgery_Schwartz. this enormous opportunity to collaborate with basic and clinical scientists to develop the field of precision medicine and surgery this century.Bifunctional RNAi Technology.36 Over the last 20 years, the field has worked to define oncogene and nononcogene addic-tion, discriminate between driver and passenger genes, and appreciate the complexity of complex, robust, network interac-tions. These insights have led to a preliminary understanding of therapeutically relevant sensitivity and resistance pathway signal patterns requiring multiple target modulation. However, this knowledge has not been effectively or reproducibly clini-cally translated. Clinical response is usually far greater when a combination of single-target molecular therapy is administered. However, it must also be realized that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxic-ity has been demonstrated with combination
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Surgery_Schwartz_3445
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that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxic-ity has been demonstrated with combination small-molecule therapy. In contrast, multitargeting bifunctional short hairpin (bi-shRNA) DNA vectors are designed to limit off-target effect given the high specificity for the genes they are designed to target.Exogenously applied hairpin constructs can be designed to be incorporated into cleavage-dependent RISC or cleav-age-independent RISC complexes, or both. The concept of a bifunctional shRNA37 is to increase knockdown efficiency without loss of sequence specificity by engaging both siRNA and miRNA-like (i.e., common biogenic pathway but comple-mentary to target sequence) RISCs, thereby concurrently acti-vating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.38 Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand
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Surgery_Schwartz. that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxic-ity has been demonstrated with combination small-molecule therapy. In contrast, multitargeting bifunctional short hairpin (bi-shRNA) DNA vectors are designed to limit off-target effect given the high specificity for the genes they are designed to target.Exogenously applied hairpin constructs can be designed to be incorporated into cleavage-dependent RISC or cleav-age-independent RISC complexes, or both. The concept of a bifunctional shRNA37 is to increase knockdown efficiency without loss of sequence specificity by engaging both siRNA and miRNA-like (i.e., common biogenic pathway but comple-mentary to target sequence) RISCs, thereby concurrently acti-vating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.38 Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand
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Surgery_Schwartz_3446
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concurrently acti-vating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.38 Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand cleavage and a second partial mismatched stem sequence for cleavage-independent passenger strand departure. Thus, functionality of the effectors is set by programmed passenger strand guided RISC loading rather than Ago subset distribution in the cancer cell. Both component Ago2 and Ago (1, 2, 4 ± 3) RNAi moieties are fully complementary to the mRNA target sequence. Preliminary data indicate reduced “off-target effects” by shRNA compared with target-identical siRNAs. More than two mismatches in sequences within the target region drasti-cally reduce knockdown effect to undetectable levels (unpub-lished results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate
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Surgery_Schwartz. concurrently acti-vating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.38 Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand cleavage and a second partial mismatched stem sequence for cleavage-independent passenger strand departure. Thus, functionality of the effectors is set by programmed passenger strand guided RISC loading rather than Ago subset distribution in the cancer cell. Both component Ago2 and Ago (1, 2, 4 ± 3) RNAi moieties are fully complementary to the mRNA target sequence. Preliminary data indicate reduced “off-target effects” by shRNA compared with target-identical siRNAs. More than two mismatches in sequences within the target region drasti-cally reduce knockdown effect to undetectable levels (unpub-lished results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate
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Surgery_Schwartz_3447
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results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate persistent susceptibility to shRNA-mediated gene 5knockdown despite recent evidence of reduced Dicer expression in human cancer cells.39The first clinical experience with the bi-shRNA platform involved the ex vivo knockdown of furin, a Ca2+-dependent, nonredundant proprotein convertase that is essential for pro-teolytic maturational processing of immunosuppressive TGF-β isoforms (β1 and β2). An autologous whole-cell cancer vac-cine, FANG™ (furin-knockdown and GMCSF-augmented),40 was produced based on a dual function immunosensitization principle of augmenting tumor antigen expression, presentation, and processing via granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are
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Surgery_Schwartz. results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate persistent susceptibility to shRNA-mediated gene 5knockdown despite recent evidence of reduced Dicer expression in human cancer cells.39The first clinical experience with the bi-shRNA platform involved the ex vivo knockdown of furin, a Ca2+-dependent, nonredundant proprotein convertase that is essential for pro-teolytic maturational processing of immunosuppressive TGF-β isoforms (β1 and β2). An autologous whole-cell cancer vac-cine, FANG™ (furin-knockdown and GMCSF-augmented),40 was produced based on a dual function immunosensitization principle of augmenting tumor antigen expression, presentation, and processing via granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are
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Surgery_Schwartz_3448
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and processing via granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are transfected with the GMCSF/bi-shRNAfurin (FANG) expression plasmid via electroporation. A phase I clinical trial (BB-IND 14205) involving 52 cancer patients was recently completed. Results demonstrated better than 90% knockdown of the bi-shRNA target, furin, and better than 90% knockdown of furin-regulated proteins TGF-β1 and TGF-β2, thereby confirming the mechanistic expectation of this novel RNAi platform. Moreover, predicted extensive GMCSF expres-sion verified our ability to successfully construct multi-cassette vectors with good manufacturing practice techniques fulfilling Food and Drug Administration requirements for clinical testing.Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified.
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Surgery_Schwartz. and processing via granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are transfected with the GMCSF/bi-shRNAfurin (FANG) expression plasmid via electroporation. A phase I clinical trial (BB-IND 14205) involving 52 cancer patients was recently completed. Results demonstrated better than 90% knockdown of the bi-shRNA target, furin, and better than 90% knockdown of furin-regulated proteins TGF-β1 and TGF-β2, thereby confirming the mechanistic expectation of this novel RNAi platform. Moreover, predicted extensive GMCSF expres-sion verified our ability to successfully construct multi-cassette vectors with good manufacturing practice techniques fulfilling Food and Drug Administration requirements for clinical testing.Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified.
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requirements for clinical testing.Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified. Median survival of the FANG™-treated patients from time of procurement was 554 days and has not been reached from time of treatment. Expected survival of similar patients is historically less than 1 year. Sequential enzyme-linked immunosorbent spot (ELISPOT) analysis revealed a dramatic and significant increase in immune response from baseline to month 4 in half of the FANG™-treated patients. Comparison of survival between ELISPOT-positive and ELISPOT-negative patients demonstrated a statistically significant increase in survival from time of procurement (P = .045) and time of treatment (P = .025).These phase 1 study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clini-cal functionality of a multitargeting (dual) DNA expression vec-tor. Further utilization of
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Surgery_Schwartz. requirements for clinical testing.Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified. Median survival of the FANG™-treated patients from time of procurement was 554 days and has not been reached from time of treatment. Expected survival of similar patients is historically less than 1 year. Sequential enzyme-linked immunosorbent spot (ELISPOT) analysis revealed a dramatic and significant increase in immune response from baseline to month 4 in half of the FANG™-treated patients. Comparison of survival between ELISPOT-positive and ELISPOT-negative patients demonstrated a statistically significant increase in survival from time of procurement (P = .045) and time of treatment (P = .025).These phase 1 study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clini-cal functionality of a multitargeting (dual) DNA expression vec-tor. Further utilization of
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Surgery_Schwartz_3450
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Surgery_Schwartz
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1 study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clini-cal functionality of a multitargeting (dual) DNA expression vec-tor. Further utilization of bi-shRNAi technology is under way clinically (targeting STMN1, a microtubule modulation criti-cal to cancer program) and preclinically targeting PDX141 (an oncogene-like transcription factor for pancreatic embryogenesis using nonviral nanoparticle delivery mechanisms).42Precision Medicine and Surgery43Genes determine our susceptibility to diseases and direct our body’s response to medicine. Because an individual’s genes dif-fer from those of another, the determination of each individual’s genome has the potential to improve the predication, prevention, and treatment of disease. Sequencing of individual genomes holds the key to realize this revolution called precision medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to
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Surgery_Schwartz. 1 study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clini-cal functionality of a multitargeting (dual) DNA expression vec-tor. Further utilization of bi-shRNAi technology is under way clinically (targeting STMN1, a microtubule modulation criti-cal to cancer program) and preclinically targeting PDX141 (an oncogene-like transcription factor for pancreatic embryogenesis using nonviral nanoparticle delivery mechanisms).42Precision Medicine and Surgery43Genes determine our susceptibility to diseases and direct our body’s response to medicine. Because an individual’s genes dif-fer from those of another, the determination of each individual’s genome has the potential to improve the predication, prevention, and treatment of disease. Sequencing of individual genomes holds the key to realize this revolution called precision medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to
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Surgery_Schwartz_3451
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genomes holds the key to realize this revolution called precision medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to reduce the time and cost so that genome sequencing can be affordable within healthcare systems. The goal of precision medicine and surgery is to identify the gene variations in each individual and to target the specific gene variations causing the disease by choosing personalized treatments that effectively work in association with the individual’s genomic profile. The importance of surgeons in this transformational field of Brunicardi_Ch15_p0479-p0510.indd 50518/02/19 11:12 AM 506BASIC CONSIDERATIONSPART Ibiomedical science is that surgeons have access to the diseased tissues on a daily basis. Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues and determine how this information can improve the outcomes of
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Surgery_Schwartz. genomes holds the key to realize this revolution called precision medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to reduce the time and cost so that genome sequencing can be affordable within healthcare systems. The goal of precision medicine and surgery is to identify the gene variations in each individual and to target the specific gene variations causing the disease by choosing personalized treatments that effectively work in association with the individual’s genomic profile. The importance of surgeons in this transformational field of Brunicardi_Ch15_p0479-p0510.indd 50518/02/19 11:12 AM 506BASIC CONSIDERATIONSPART Ibiomedical science is that surgeons have access to the diseased tissues on a daily basis. Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues and determine how this information can improve the outcomes of
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Surgery_Schwartz_3452
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Surgery_Schwartz
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Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues and determine how this information can improve the outcomes of surgery, i.e., precision surgery. These discovery studies are rapidly leading to the uncovering of mutations and SNPs that are the underlying cause of an individual’s disease and ultimately lead to targeted thera-pies. Although precision medicine and surgery holds the poten-tial to revolutionize the practice of modern medicine, there currently exists a gap between our ability to sequence any given individual’s genome and how clinicians can apply this informa-tion to guide care. There is a rapidly growing list of single genes that are currently guiding care, and these genes are listed as type 1 precision genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrec-tomy, respectively; however, the great
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Surgery_Schwartz. Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues and determine how this information can improve the outcomes of surgery, i.e., precision surgery. These discovery studies are rapidly leading to the uncovering of mutations and SNPs that are the underlying cause of an individual’s disease and ultimately lead to targeted thera-pies. Although precision medicine and surgery holds the poten-tial to revolutionize the practice of modern medicine, there currently exists a gap between our ability to sequence any given individual’s genome and how clinicians can apply this informa-tion to guide care. There is a rapidly growing list of single genes that are currently guiding care, and these genes are listed as type 1 precision genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrec-tomy, respectively; however, the great
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Surgery_Schwartz_3453
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1 precision genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrec-tomy, respectively; however, the great challenge before the sci-entific and medical community this century is to learn to use the entire genome to guide precision care.Targeted Genome Editing Using the CRISPR-Cas9 SystemConventional genetic manipulations have proven their value in biomedical research. Researchers today depend on the manipu-lation of genetic materials in cells or in animal models in almost every project they work on. These genetic manipulation tech-niques (see “Genetic Manipulations”), though sufficient for gen-eral research purposes, suffer from disadvantages. Transfection of target genes into cells is quick and specific, but nonnative. RNA interference (RNAi) is easy to perform and targets native genes, but RNAi never fully eliminates the target gene, and off-target effects are commonly seen using RNAi. Gene
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Surgery_Schwartz. 1 precision genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrec-tomy, respectively; however, the great challenge before the sci-entific and medical community this century is to learn to use the entire genome to guide precision care.Targeted Genome Editing Using the CRISPR-Cas9 SystemConventional genetic manipulations have proven their value in biomedical research. Researchers today depend on the manipu-lation of genetic materials in cells or in animal models in almost every project they work on. These genetic manipulation tech-niques (see “Genetic Manipulations”), though sufficient for gen-eral research purposes, suffer from disadvantages. Transfection of target genes into cells is quick and specific, but nonnative. RNA interference (RNAi) is easy to perform and targets native genes, but RNAi never fully eliminates the target gene, and off-target effects are commonly seen using RNAi. Gene
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Surgery_Schwartz_3454
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Surgery_Schwartz
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but nonnative. RNA interference (RNAi) is easy to perform and targets native genes, but RNAi never fully eliminates the target gene, and off-target effects are commonly seen using RNAi. Gene knockout mice provide an ideal platform to study native genes with clean background, but conventional knockout methods are time-consuming and costly.An entirely new gene-editing method now known as the CRISPR-Cas9 system has emerged since 201344-46 and has quickly gained popularity among biologists for gene edit-ing. This new method is easy to perform, can work specifi-cally on the desired gene or DNA sequence, and can generate gene knockout, knock-in, point mutation, or the insertion of an epitope tag in almost any cell line or animal models with high efficiencies.CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.47 It is a region on the genomic DNA first discovered in the microbes as an adapted immune system against exogenous DNA. A typical CRISPR region contains a
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Surgery_Schwartz. but nonnative. RNA interference (RNAi) is easy to perform and targets native genes, but RNAi never fully eliminates the target gene, and off-target effects are commonly seen using RNAi. Gene knockout mice provide an ideal platform to study native genes with clean background, but conventional knockout methods are time-consuming and costly.An entirely new gene-editing method now known as the CRISPR-Cas9 system has emerged since 201344-46 and has quickly gained popularity among biologists for gene edit-ing. This new method is easy to perform, can work specifi-cally on the desired gene or DNA sequence, and can generate gene knockout, knock-in, point mutation, or the insertion of an epitope tag in almost any cell line or animal models with high efficiencies.CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.47 It is a region on the genomic DNA first discovered in the microbes as an adapted immune system against exogenous DNA. A typical CRISPR region contains a
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Surgery_Schwartz_3455
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Interspaced Short Palindromic Repeats.47 It is a region on the genomic DNA first discovered in the microbes as an adapted immune system against exogenous DNA. A typical CRISPR region contains a cluster of short (21–48 bp) DNA repeats (ranging from 2 to hundreds) interspaced by nonrepetitive sequences called spacers.47 Within a CRISPR region, while each spacer has its unique sequence, the sequence of the repeats is highly conserved. Several genes, called the CRISPR-associated (Cas) genes, are almost always found directly flanking the CRISPR region.Extensive studies in the past decade revealed the function of the CRISPR-cas system in DNA-interfering. When bacte-ria or archaea carrying the CRISPR-cas system are invaded by phage or plasmid DNA, a new spacer can be added to the CRISPR region with its sequence identical to the “proto-spacer,” a fragment of the invading DNA.47 It was found that the proto-spacer must be followed by a recognition sequence (NGG in the case of Cas9) called
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Surgery_Schwartz. Interspaced Short Palindromic Repeats.47 It is a region on the genomic DNA first discovered in the microbes as an adapted immune system against exogenous DNA. A typical CRISPR region contains a cluster of short (21–48 bp) DNA repeats (ranging from 2 to hundreds) interspaced by nonrepetitive sequences called spacers.47 Within a CRISPR region, while each spacer has its unique sequence, the sequence of the repeats is highly conserved. Several genes, called the CRISPR-associated (Cas) genes, are almost always found directly flanking the CRISPR region.Extensive studies in the past decade revealed the function of the CRISPR-cas system in DNA-interfering. When bacte-ria or archaea carrying the CRISPR-cas system are invaded by phage or plasmid DNA, a new spacer can be added to the CRISPR region with its sequence identical to the “proto-spacer,” a fragment of the invading DNA.47 It was found that the proto-spacer must be followed by a recognition sequence (NGG in the case of Cas9) called
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Surgery_Schwartz_3456
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with its sequence identical to the “proto-spacer,” a fragment of the invading DNA.47 It was found that the proto-spacer must be followed by a recognition sequence (NGG in the case of Cas9) called proto-spacer associated motif 67(PAM).46 With constitutive transcription, the CRISPR region is transcribed as mRNA, and cut by Cas proteins to generate RNA fragments minimally containing one spacer and parts of the repetitive sequence.47 This fragment associates with target DNA through Watson-Crick base-pairing and directs the cutting of target DNA by Cas proteins with nuclease activity.Several CRISPR-Cas systems have been characterized, with a variety of Cas proteins found in these systems. Cas9 in the type II system from Streptococcus pyogenes is the most commonly used Cas for gene editing.46,48 Cas9 contains one RuvC-like nuclease domain near its N-terminal and one HNH-like nuclease domain in the middle of the protein. The RuvC-like domain cuts the proto-spacer strand (the strand with
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Surgery_Schwartz. with its sequence identical to the “proto-spacer,” a fragment of the invading DNA.47 It was found that the proto-spacer must be followed by a recognition sequence (NGG in the case of Cas9) called proto-spacer associated motif 67(PAM).46 With constitutive transcription, the CRISPR region is transcribed as mRNA, and cut by Cas proteins to generate RNA fragments minimally containing one spacer and parts of the repetitive sequence.47 This fragment associates with target DNA through Watson-Crick base-pairing and directs the cutting of target DNA by Cas proteins with nuclease activity.Several CRISPR-Cas systems have been characterized, with a variety of Cas proteins found in these systems. Cas9 in the type II system from Streptococcus pyogenes is the most commonly used Cas for gene editing.46,48 Cas9 contains one RuvC-like nuclease domain near its N-terminal and one HNH-like nuclease domain in the middle of the protein. The RuvC-like domain cuts the proto-spacer strand (the strand with
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Cas9 contains one RuvC-like nuclease domain near its N-terminal and one HNH-like nuclease domain in the middle of the protein. The RuvC-like domain cuts the proto-spacer strand (the strand with PAM), and the HNH-like domain cuts the strand pairing with the spacer,49 resulting in a double-strand break (DSB) on the target DNA. It is known that Cas9 cuts a blunt end 3 bp 5’ to the PAM sequence.46 The specificity provided by the spacer, or so-called “guide RNA,” and the ability of Cas9 to cut double-stranded DNA means that this system can specifically target anywhere in a genome with a known sequence.CRISPR-Cas9–Guided Gene Editing. The CRISPR-Cas9 sys-tem was made suitable for gene editing in mammalian cell lines and in animal models through several years of optimization. The key concept for CRISPR-Cas9–mediated gene editing is to introduce DNA strand break and let the cell repair the break. Through the repair process, sequence deletions, insertions, and mutations can be applied to the
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Surgery_Schwartz. Cas9 contains one RuvC-like nuclease domain near its N-terminal and one HNH-like nuclease domain in the middle of the protein. The RuvC-like domain cuts the proto-spacer strand (the strand with PAM), and the HNH-like domain cuts the strand pairing with the spacer,49 resulting in a double-strand break (DSB) on the target DNA. It is known that Cas9 cuts a blunt end 3 bp 5’ to the PAM sequence.46 The specificity provided by the spacer, or so-called “guide RNA,” and the ability of Cas9 to cut double-stranded DNA means that this system can specifically target anywhere in a genome with a known sequence.CRISPR-Cas9–Guided Gene Editing. The CRISPR-Cas9 sys-tem was made suitable for gene editing in mammalian cell lines and in animal models through several years of optimization. The key concept for CRISPR-Cas9–mediated gene editing is to introduce DNA strand break and let the cell repair the break. Through the repair process, sequence deletions, insertions, and mutations can be applied to the
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CRISPR-Cas9–mediated gene editing is to introduce DNA strand break and let the cell repair the break. Through the repair process, sequence deletions, insertions, and mutations can be applied to the target gene.Two DNA repair pathways are utilized in CRISPR-Cas9–mediated gene editing: the nonhomologous end-joining (NHEJ) pathway and the homology-directed repair (HDR).49 When a homologous repair template is unavailable, the NHEJ path-way joins the ends of the DSB together, usually with random insertion/deletion (indel) mutations. Such mutations within an open reading frame can cause frameshift and/or premature stop codons. When a repair template is available, however, the cell may choose HDR and repair the DSB through pairing with the template. The HDR pathway can introduce longer insertion/ deletion mutations than NHEJ and can specify the mutated sequence (by contrast, NHEJ creates random mutations). However, HDR is usually only active in dividing cells and is of a lower efficiency
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Surgery_Schwartz. CRISPR-Cas9–mediated gene editing is to introduce DNA strand break and let the cell repair the break. Through the repair process, sequence deletions, insertions, and mutations can be applied to the target gene.Two DNA repair pathways are utilized in CRISPR-Cas9–mediated gene editing: the nonhomologous end-joining (NHEJ) pathway and the homology-directed repair (HDR).49 When a homologous repair template is unavailable, the NHEJ path-way joins the ends of the DSB together, usually with random insertion/deletion (indel) mutations. Such mutations within an open reading frame can cause frameshift and/or premature stop codons. When a repair template is available, however, the cell may choose HDR and repair the DSB through pairing with the template. The HDR pathway can introduce longer insertion/ deletion mutations than NHEJ and can specify the mutated sequence (by contrast, NHEJ creates random mutations). However, HDR is usually only active in dividing cells and is of a lower efficiency
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deletion mutations than NHEJ and can specify the mutated sequence (by contrast, NHEJ creates random mutations). However, HDR is usually only active in dividing cells and is of a lower efficiency than NHEJ. Its efficiency also depends on gene loca-tion, cell type, and the repair template.50 Therefore, the choice of the pathway depends on the need of the outcome: to simply create a gene knockout, NHEJ is much simpler and highly effi-cient; to achieve precise gene editing (introduce a specific muta-tion, add or delete a specific sequence), HDR must be used. The two pathways have similar protocols, but they differ in certain details in experimental designs.Gene Editing Through NHEJ Tools for NHEJ-mediated gene editing can be incorporated onto one simple plasmid, including a single-guide RNA (sgRNA) sequence containing the guide RNA, a U6 promoter driving the expression of the sgRNA, and an expression cassette including codon-optimized Cas9 fused with nuclear localization sequences (NLS)
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Surgery_Schwartz. deletion mutations than NHEJ and can specify the mutated sequence (by contrast, NHEJ creates random mutations). However, HDR is usually only active in dividing cells and is of a lower efficiency than NHEJ. Its efficiency also depends on gene loca-tion, cell type, and the repair template.50 Therefore, the choice of the pathway depends on the need of the outcome: to simply create a gene knockout, NHEJ is much simpler and highly effi-cient; to achieve precise gene editing (introduce a specific muta-tion, add or delete a specific sequence), HDR must be used. The two pathways have similar protocols, but they differ in certain details in experimental designs.Gene Editing Through NHEJ Tools for NHEJ-mediated gene editing can be incorporated onto one simple plasmid, including a single-guide RNA (sgRNA) sequence containing the guide RNA, a U6 promoter driving the expression of the sgRNA, and an expression cassette including codon-optimized Cas9 fused with nuclear localization sequences (NLS)
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(sgRNA) sequence containing the guide RNA, a U6 promoter driving the expression of the sgRNA, and an expression cassette including codon-optimized Cas9 fused with nuclear localization sequences (NLS) and an optional selection marker (puromycin resistance or a GFP).46 NHEJ is usually used to knock out a target gene (Fig. 15-24). Generally, a 20 bp sequence preceding a PAM sequence (NGG for Cas9) is selected as the guide RNA sequence. This sequence can be chosen within the first couple of exons through running online search engines against the target genome to minimize off-target probabilities. The guide RNA sequence is then inserted into the Brunicardi_Ch15_p0479-p0510.indd 50618/02/19 11:12 AM 507MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Figure 15-25. CRISPR-Cas9–mediated gene editing through NHEJ or HDR. The Cas9 protein cleaves target DNA through its RuvC-like and HNH-like domains, guided by the sgRNA. The cell repairs the double-strand
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Surgery_Schwartz. (sgRNA) sequence containing the guide RNA, a U6 promoter driving the expression of the sgRNA, and an expression cassette including codon-optimized Cas9 fused with nuclear localization sequences (NLS) and an optional selection marker (puromycin resistance or a GFP).46 NHEJ is usually used to knock out a target gene (Fig. 15-24). Generally, a 20 bp sequence preceding a PAM sequence (NGG for Cas9) is selected as the guide RNA sequence. This sequence can be chosen within the first couple of exons through running online search engines against the target genome to minimize off-target probabilities. The guide RNA sequence is then inserted into the Brunicardi_Ch15_p0479-p0510.indd 50618/02/19 11:12 AM 507MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Figure 15-25. CRISPR-Cas9–mediated gene editing through NHEJ or HDR. The Cas9 protein cleaves target DNA through its RuvC-like and HNH-like domains, guided by the sgRNA. The cell repairs the double-strand
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15-25. CRISPR-Cas9–mediated gene editing through NHEJ or HDR. The Cas9 protein cleaves target DNA through its RuvC-like and HNH-like domains, guided by the sgRNA. The cell repairs the double-strand break through either NHEJ or HDR. In NHEJ, the broken ends are recognized, bound, and tethered by end-binding protein complexes. The ends are then processed and ligated but may result in ran-dom insertion/deletion (experimentally, deletions are more commonly seen) mutations at the break site. The repair process in HDR, however, generates no random error due to the presence of a homologous repair template. During the repair, the broken strands find the homologous template and proceed with DNA synthesis using the template. This results in a repaired DNA that has the same sequence as the repair template. Therefore, point mutations, insertions, or deletions carried on the template will be inherited by the repaired DNA and thus achieve precise gene editing.designed site within the sgRNA, and the
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Surgery_Schwartz. 15-25. CRISPR-Cas9–mediated gene editing through NHEJ or HDR. The Cas9 protein cleaves target DNA through its RuvC-like and HNH-like domains, guided by the sgRNA. The cell repairs the double-strand break through either NHEJ or HDR. In NHEJ, the broken ends are recognized, bound, and tethered by end-binding protein complexes. The ends are then processed and ligated but may result in ran-dom insertion/deletion (experimentally, deletions are more commonly seen) mutations at the break site. The repair process in HDR, however, generates no random error due to the presence of a homologous repair template. During the repair, the broken strands find the homologous template and proceed with DNA synthesis using the template. This results in a repaired DNA that has the same sequence as the repair template. Therefore, point mutations, insertions, or deletions carried on the template will be inherited by the repaired DNA and thus achieve precise gene editing.designed site within the sgRNA, and the
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Therefore, point mutations, insertions, or deletions carried on the template will be inherited by the repaired DNA and thus achieve precise gene editing.designed site within the sgRNA, and the constructed plasmid is transfected into target cells. Expressed Cas9 proteins would associate with expressed sgRNAs to mediate DSB directed by the guide RNA sequence. For cell lines, pure mutant clones can be generated by separating single colonies. Knockout mutations can be confirmed through PCR amplification of the target region and sequencing. If antibodies against the target protein are avail-able, western blotting can be used as a supplemental method to DNA sequencing results to confirm the knockout of the gene.Gene Editing Through HDR HDR requires a homologous tem-plate to repair the DSB. Therefore, apart from the guide RNA, another sequence homologous to the flanking regions of the DSB needs to be present (Fig. 15-25). The homologous sequence can carry insertions, deletions, and mutations
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Surgery_Schwartz. Therefore, point mutations, insertions, or deletions carried on the template will be inherited by the repaired DNA and thus achieve precise gene editing.designed site within the sgRNA, and the constructed plasmid is transfected into target cells. Expressed Cas9 proteins would associate with expressed sgRNAs to mediate DSB directed by the guide RNA sequence. For cell lines, pure mutant clones can be generated by separating single colonies. Knockout mutations can be confirmed through PCR amplification of the target region and sequencing. If antibodies against the target protein are avail-able, western blotting can be used as a supplemental method to DNA sequencing results to confirm the knockout of the gene.Gene Editing Through HDR HDR requires a homologous tem-plate to repair the DSB. Therefore, apart from the guide RNA, another sequence homologous to the flanking regions of the DSB needs to be present (Fig. 15-25). The homologous sequence can carry insertions, deletions, and mutations
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apart from the guide RNA, another sequence homologous to the flanking regions of the DSB needs to be present (Fig. 15-25). The homologous sequence can carry insertions, deletions, and mutations to replace the sequence at the DSB, thus achieving precise gene editing on the target. The homologous sequence can be introduced into the cell either as a template on a plasmid or as a single-stranded oligonucleotide.46 After the induction of the DSB, the homologous template pairs with the flanking regions of the DSB and serves as the template for the repair of the break site. HDR happens at lower efficiency than NHEJ and therefore is of lower success rate than NHEJ-mediated gene editing. Therefore, HDR is only recommended when precise mutations are desired. Fast screen of positive mutant clones can be achieved by incorporating restriction enzyme cutting sites within the homologous template.Reducing Off-Target Effects Using Cas9 Nickase The CRISPR-Cas9 system uses a 20 bp guide RNA for sequence
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Surgery_Schwartz. apart from the guide RNA, another sequence homologous to the flanking regions of the DSB needs to be present (Fig. 15-25). The homologous sequence can carry insertions, deletions, and mutations to replace the sequence at the DSB, thus achieving precise gene editing on the target. The homologous sequence can be introduced into the cell either as a template on a plasmid or as a single-stranded oligonucleotide.46 After the induction of the DSB, the homologous template pairs with the flanking regions of the DSB and serves as the template for the repair of the break site. HDR happens at lower efficiency than NHEJ and therefore is of lower success rate than NHEJ-mediated gene editing. Therefore, HDR is only recommended when precise mutations are desired. Fast screen of positive mutant clones can be achieved by incorporating restriction enzyme cutting sites within the homologous template.Reducing Off-Target Effects Using Cas9 Nickase The CRISPR-Cas9 system uses a 20 bp guide RNA for sequence
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be achieved by incorporating restriction enzyme cutting sites within the homologous template.Reducing Off-Target Effects Using Cas9 Nickase The CRISPR-Cas9 system uses a 20 bp guide RNA for sequence recognition. Due to the similar length of recognition sequence to RNAi techniques, CRISPR-Cas9 system also suffers from off-target effects. Because CRISPR-Cas9 requires a PAM site directly following the guide RNA sequence, one way to reduce off-targets is to run through online databases against the genome of the target cell to select a target sequence with the least number of possible off-targets (CRISPR-Cas9 system tolerates no more than three mismatches). On the other hand, a mutant version of the Cas9 called Cas9 nickase can be used to minimize the risk of off-targets.As mentioned, Cas9 contains one RuvC-like and one HNH-like nuclease domain, each responsible for cutting one strand.49 The D10A mutant Cas9 (Cas9 nickase) lacks the activ-ity of the RuvC-like domain, leaving the
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Surgery_Schwartz. be achieved by incorporating restriction enzyme cutting sites within the homologous template.Reducing Off-Target Effects Using Cas9 Nickase The CRISPR-Cas9 system uses a 20 bp guide RNA for sequence recognition. Due to the similar length of recognition sequence to RNAi techniques, CRISPR-Cas9 system also suffers from off-target effects. Because CRISPR-Cas9 requires a PAM site directly following the guide RNA sequence, one way to reduce off-targets is to run through online databases against the genome of the target cell to select a target sequence with the least number of possible off-targets (CRISPR-Cas9 system tolerates no more than three mismatches). On the other hand, a mutant version of the Cas9 called Cas9 nickase can be used to minimize the risk of off-targets.As mentioned, Cas9 contains one RuvC-like and one HNH-like nuclease domain, each responsible for cutting one strand.49 The D10A mutant Cas9 (Cas9 nickase) lacks the activ-ity of the RuvC-like domain, leaving the
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Cas9 contains one RuvC-like and one HNH-like nuclease domain, each responsible for cutting one strand.49 The D10A mutant Cas9 (Cas9 nickase) lacks the activ-ity of the RuvC-like domain, leaving the proto-spacer strand intact and a nick on the antisense strand.46 Using two prop-erly spaced (0–20 bp apart), oppositely oriented guide RNA, the Cas9 nickase will leave two single-strand nicks on both strands in close proximity, creating a double-strand break with 5’ overhangs on both strands. This leads to NHEJ or HDR at this breaking site, while other off-target single-strand nicks will be repaired without impact. This strategy doubles the number of base pairs required for site recognition, reducing off-target possibilities almost to zero (from about 1 off-target in 30 million bp to 1 in 1000 trillion bp).Application of the CRISPR-Cas9 System in Biomedical Sciences The biggest advantage of the CRISPR-Cas9 system is its ability to edit genes in almost any cell type and any ani-mal model
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Surgery_Schwartz. Cas9 contains one RuvC-like and one HNH-like nuclease domain, each responsible for cutting one strand.49 The D10A mutant Cas9 (Cas9 nickase) lacks the activ-ity of the RuvC-like domain, leaving the proto-spacer strand intact and a nick on the antisense strand.46 Using two prop-erly spaced (0–20 bp apart), oppositely oriented guide RNA, the Cas9 nickase will leave two single-strand nicks on both strands in close proximity, creating a double-strand break with 5’ overhangs on both strands. This leads to NHEJ or HDR at this breaking site, while other off-target single-strand nicks will be repaired without impact. This strategy doubles the number of base pairs required for site recognition, reducing off-target possibilities almost to zero (from about 1 off-target in 30 million bp to 1 in 1000 trillion bp).Application of the CRISPR-Cas9 System in Biomedical Sciences The biggest advantage of the CRISPR-Cas9 system is its ability to edit genes in almost any cell type and any ani-mal model
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trillion bp).Application of the CRISPR-Cas9 System in Biomedical Sciences The biggest advantage of the CRISPR-Cas9 system is its ability to edit genes in almost any cell type and any ani-mal model with high efficiency and accuracy. Plus, it is easy to design and easy to use. Within a couple of years, success-ful gene editing in C. elegans,51 zebrafish,52 fruit fly,53 mouse,54 dogs,55 and even nonhuman primates56 was achieved. CRISPR-Cas9 was also reported to be successful in a variety of cell types including stem cells.Currently, CRISPR-Cas9 is most used for editing single genes, through gene knockout, gene mutation, or the addition of an epitope tag to a native gene, for functional characterization of the gene of interest. For example, oncogenes or tumor suppres-sor genes can be knocked out to identify the causative gene for a particular cancer type; point mutations in functional domains homologous templateprecise editingNHEJ5’3’5’3’5’3’5’3’5’3’3’5’3’5’3’5’3’5’3’5’3’Cas9 RuvC
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Surgery_Schwartz. trillion bp).Application of the CRISPR-Cas9 System in Biomedical Sciences The biggest advantage of the CRISPR-Cas9 system is its ability to edit genes in almost any cell type and any ani-mal model with high efficiency and accuracy. Plus, it is easy to design and easy to use. Within a couple of years, success-ful gene editing in C. elegans,51 zebrafish,52 fruit fly,53 mouse,54 dogs,55 and even nonhuman primates56 was achieved. CRISPR-Cas9 was also reported to be successful in a variety of cell types including stem cells.Currently, CRISPR-Cas9 is most used for editing single genes, through gene knockout, gene mutation, or the addition of an epitope tag to a native gene, for functional characterization of the gene of interest. For example, oncogenes or tumor suppres-sor genes can be knocked out to identify the causative gene for a particular cancer type; point mutations in functional domains homologous templateprecise editingNHEJ5’3’5’3’5’3’5’3’5’3’3’5’3’5’3’5’3’5’3’5’3’Cas9 RuvC
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out to identify the causative gene for a particular cancer type; point mutations in functional domains homologous templateprecise editingNHEJ5’3’5’3’5’3’5’3’5’3’3’5’3’5’3’5’3’5’3’5’3’Cas9 RuvC domainHNHdomainPAMsgRNA5’5’3’HDRRandom insertion/deletionBrunicardi_Ch15_p0479-p0510.indd 50718/02/19 11:12 AM 508BASIC CONSIDERATIONSPART Imay illustrate the mechanism of action of a protein; for proteins without available antibodies, epitope tags can be inserted onto the native gene for the detection of the native protein.Apart from single gene editing, CRISPR-Cas9 can be used for large scale loss-of-function gene screen. Multiple lentiviral guide RNA libraries have been established covering the whole human/mouse genome or particular subsets.Owing to its ability to bind to specific nucleotide sequences, CRISPR-Cas9 has also been used for non–gene-editing purposes. To achieve this, both the RuvC-like and the HNH-like domains are mutated to give a catalytically inactive Cas9 (dCas9).
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Surgery_Schwartz. out to identify the causative gene for a particular cancer type; point mutations in functional domains homologous templateprecise editingNHEJ5’3’5’3’5’3’5’3’5’3’3’5’3’5’3’5’3’5’3’5’3’Cas9 RuvC domainHNHdomainPAMsgRNA5’5’3’HDRRandom insertion/deletionBrunicardi_Ch15_p0479-p0510.indd 50718/02/19 11:12 AM 508BASIC CONSIDERATIONSPART Imay illustrate the mechanism of action of a protein; for proteins without available antibodies, epitope tags can be inserted onto the native gene for the detection of the native protein.Apart from single gene editing, CRISPR-Cas9 can be used for large scale loss-of-function gene screen. Multiple lentiviral guide RNA libraries have been established covering the whole human/mouse genome or particular subsets.Owing to its ability to bind to specific nucleotide sequences, CRISPR-Cas9 has also been used for non–gene-editing purposes. To achieve this, both the RuvC-like and the HNH-like domains are mutated to give a catalytically inactive Cas9 (dCas9).
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sequences, CRISPR-Cas9 has also been used for non–gene-editing purposes. To achieve this, both the RuvC-like and the HNH-like domains are mutated to give a catalytically inactive Cas9 (dCas9). Directed by guide RNA, this dCas9 can bind to particu-lar genes to reversibly suppress or activate gene transcription by the fusion of transcription activators or suppressors with dCas9. Epigenetic modulators (e.g., DNA methylase) can also be fused with dCas9 to achieve controlled epigenetic modulations. More-over, dCas9 can also be fused with fluorescent markers such as GFP to track a particular DNA region in live cells.The most exciting potential application of the CRISPR-Cas9 system is perhaps the correction or modification of dis-ease-causing genes in human embryos or in human patients to eradicate disease-causing genes. However, extension of this application may lead to the creation of the so called “perfect human,” hence raising huge ethical concerns and controversies.57 Because of such
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Surgery_Schwartz. sequences, CRISPR-Cas9 has also been used for non–gene-editing purposes. To achieve this, both the RuvC-like and the HNH-like domains are mutated to give a catalytically inactive Cas9 (dCas9). Directed by guide RNA, this dCas9 can bind to particu-lar genes to reversibly suppress or activate gene transcription by the fusion of transcription activators or suppressors with dCas9. Epigenetic modulators (e.g., DNA methylase) can also be fused with dCas9 to achieve controlled epigenetic modulations. More-over, dCas9 can also be fused with fluorescent markers such as GFP to track a particular DNA region in live cells.The most exciting potential application of the CRISPR-Cas9 system is perhaps the correction or modification of dis-ease-causing genes in human embryos or in human patients to eradicate disease-causing genes. However, extension of this application may lead to the creation of the so called “perfect human,” hence raising huge ethical concerns and controversies.57 Because of such
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disease-causing genes. However, extension of this application may lead to the creation of the so called “perfect human,” hence raising huge ethical concerns and controversies.57 Because of such ethical considerations, gene editing in human embryos should be cautiously conducted. Nonetheless, reports have shown the great potential of this powerful gene-editing technique in correcting gene mutations.58-60In conclusion, the CRISPR-Cas9 system is the most pow-erful gene-editing system characterized so far, showing its strong ability to edit genes efficiently and precisely. Its applica-tions greatly benefit biomedical researches and, with a solution to ethical issues, can greatly benefit clinical medicine as well.REFERENCESEntries highlighted in bright blue are key references. 1. Watson JD, Crick FH. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature. 1953;171(4356):737-738. 2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th ed.
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Surgery_Schwartz. disease-causing genes. However, extension of this application may lead to the creation of the so called “perfect human,” hence raising huge ethical concerns and controversies.57 Because of such ethical considerations, gene editing in human embryos should be cautiously conducted. Nonetheless, reports have shown the great potential of this powerful gene-editing technique in correcting gene mutations.58-60In conclusion, the CRISPR-Cas9 system is the most pow-erful gene-editing system characterized so far, showing its strong ability to edit genes efficiently and precisely. Its applica-tions greatly benefit biomedical researches and, with a solution to ethical issues, can greatly benefit clinical medicine as well.REFERENCESEntries highlighted in bright blue are key references. 1. Watson JD, Crick FH. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature. 1953;171(4356):737-738. 2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th ed.
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FH. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature. 1953;171(4356):737-738. 2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th ed. New York: Garland Science; 2002. 3. International Human Genome Sequencing Consortium. Finish-ing the euchromatic sequence of the human genome. Nature. 2004;431(7011):931-945. 4. ENCODE Project Consortium, Birney E, Stamatoyannopou-los JA, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007;447(7146):799-816. 5. Mendel G. Versuche über Planzen-Hybriden. Verhandlungen des naturforschenden Vereines, Abhandlungen. Brünn: 4, 3, 1866. 6. Carey M, Smale ST. Transcriptional Regulation in Eukaryotes. New York: Cold Spring Harbor Laboratory Press; 2000. 7. Wolfsberg TG, Wetterstrand KA, Guyer MS, Collins FS, Baxevanis AD. A user’s guide to the human genome. Nat Genet. 2002;32(suppl):1-79. 8. U.S. Department of Energy. Genomics
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Surgery_Schwartz. FH. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature. 1953;171(4356):737-738. 2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th ed. New York: Garland Science; 2002. 3. International Human Genome Sequencing Consortium. Finish-ing the euchromatic sequence of the human genome. Nature. 2004;431(7011):931-945. 4. ENCODE Project Consortium, Birney E, Stamatoyannopou-los JA, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007;447(7146):799-816. 5. Mendel G. Versuche über Planzen-Hybriden. Verhandlungen des naturforschenden Vereines, Abhandlungen. Brünn: 4, 3, 1866. 6. Carey M, Smale ST. Transcriptional Regulation in Eukaryotes. New York: Cold Spring Harbor Laboratory Press; 2000. 7. Wolfsberg TG, Wetterstrand KA, Guyer MS, Collins FS, Baxevanis AD. A user’s guide to the human genome. Nat Genet. 2002;32(suppl):1-79. 8. U.S. Department of Energy. Genomics
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Press; 2000. 7. Wolfsberg TG, Wetterstrand KA, Guyer MS, Collins FS, Baxevanis AD. A user’s guide to the human genome. Nat Genet. 2002;32(suppl):1-79. 8. U.S. Department of Energy. Genomics and its impact on science and society: the human genome project and beyond. Published online by Human Genome Management Information System (HGMIS). Available at: http://web.ornl.gov/sci/techresources/Human_Genome/publicat/primer2001/primer11.pdf. 9. Simpson RJ. Proteins and Proteomics. New York: CSHL Press; 2003. 10. Hanash S. Disease proteomics. Nature. 2003;422(6928):226-232. 11. Ptashne M, Gann A. Genes & Signals. New York: CSHL Press; 2002. 12. Pawson T, Nash P. Assembly of cell regulatory systems through protein interaction domains. Science. 2003;300(5618): 445-452. 13. Lizcano JM, Alessi DR. The insulin signaling pathway. Curr Biol. 2002;12(7):R236-R238. 14. Feng X-H, Derynck R. Specificity and versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21:
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Surgery_Schwartz. Press; 2000. 7. Wolfsberg TG, Wetterstrand KA, Guyer MS, Collins FS, Baxevanis AD. A user’s guide to the human genome. Nat Genet. 2002;32(suppl):1-79. 8. U.S. Department of Energy. Genomics and its impact on science and society: the human genome project and beyond. Published online by Human Genome Management Information System (HGMIS). Available at: http://web.ornl.gov/sci/techresources/Human_Genome/publicat/primer2001/primer11.pdf. 9. Simpson RJ. Proteins and Proteomics. New York: CSHL Press; 2003. 10. Hanash S. Disease proteomics. Nature. 2003;422(6928):226-232. 11. Ptashne M, Gann A. Genes & Signals. New York: CSHL Press; 2002. 12. Pawson T, Nash P. Assembly of cell regulatory systems through protein interaction domains. Science. 2003;300(5618): 445-452. 13. Lizcano JM, Alessi DR. The insulin signaling pathway. Curr Biol. 2002;12(7):R236-R238. 14. Feng X-H, Derynck R. Specificity and versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21:
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Alessi DR. The insulin signaling pathway. Curr Biol. 2002;12(7):R236-R238. 14. Feng X-H, Derynck R. Specificity and versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21: 659-693. 15. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57-70. 16. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674. 17. McNeil C. Herceptin raises its sights beyond advanced breast cancer. J Natl Cancer Inst. 1998;90:882. 18. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2(5):561. 19. Kiessling AA, Anderson SC. Human Embryonic Stem Cells: An Introduction to the Science and Therapeutic Potential. Boston: Jones & Bartlett Pub; 2003. 20. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripo-tent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872. 21. Yu J, Vodyanik MA,
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Surgery_Schwartz. Alessi DR. The insulin signaling pathway. Curr Biol. 2002;12(7):R236-R238. 14. Feng X-H, Derynck R. Specificity and versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21: 659-693. 15. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57-70. 16. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674. 17. McNeil C. Herceptin raises its sights beyond advanced breast cancer. J Natl Cancer Inst. 1998;90:882. 18. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2(5):561. 19. Kiessling AA, Anderson SC. Human Embryonic Stem Cells: An Introduction to the Science and Therapeutic Potential. Boston: Jones & Bartlett Pub; 2003. 20. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripo-tent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872. 21. Yu J, Vodyanik MA,
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Bartlett Pub; 2003. 20. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripo-tent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872. 21. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917-1920. 22. Orcutt S, Nguyen T, Harring TR. Subatomic medicine and the atomic theory of disease. Transl Med. 2012;2:2. 23. Cohen SN, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA. 1973;70(11):3240-3244. 24. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. New York: Cold Spring Harbor Laboratory Press; 2012. 25. Ausubel FM, Brent R, Kingston RE, et al. Current Protocols in Molecular Biology. 3rd ed. New York: John Wiley & Sons; 1995. 26. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98(3):503. 27. Mullis K,
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Surgery_Schwartz. Bartlett Pub; 2003. 20. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripo-tent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872. 21. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917-1920. 22. Orcutt S, Nguyen T, Harring TR. Subatomic medicine and the atomic theory of disease. Transl Med. 2012;2:2. 23. Cohen SN, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA. 1973;70(11):3240-3244. 24. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. New York: Cold Spring Harbor Laboratory Press; 2012. 25. Ausubel FM, Brent R, Kingston RE, et al. Current Protocols in Molecular Biology. 3rd ed. New York: John Wiley & Sons; 1995. 26. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98(3):503. 27. Mullis K,
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Biology. 3rd ed. New York: John Wiley & Sons; 1995. 26. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98(3):503. 27. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the poly-merase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(pt 1):263-273. 28. Bowtell D, Sambrook J. DNA Microarrays: A Molecular Clon-ing Manual. 1st ed. New York: Cold Spring Harbor Laboratory Press; 2002. 29. Caruccio N. Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition. Methods Mol Biol. 2011;733:241-255. 30. Pettersson E, Lundeberg J, Ahmadian A. Generations of sequencing technologies. Genomics. 2009;93(2):105-111. 31. Biankin AV, Waddell N, Kassahn KS, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature.
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Surgery_Schwartz. Biology. 3rd ed. New York: John Wiley & Sons; 1995. 26. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98(3):503. 27. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the poly-merase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(pt 1):263-273. 28. Bowtell D, Sambrook J. DNA Microarrays: A Molecular Clon-ing Manual. 1st ed. New York: Cold Spring Harbor Laboratory Press; 2002. 29. Caruccio N. Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition. Methods Mol Biol. 2011;733:241-255. 30. Pettersson E, Lundeberg J, Ahmadian A. Generations of sequencing technologies. Genomics. 2009;93(2):105-111. 31. Biankin AV, Waddell N, Kassahn KS, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature.
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of sequencing technologies. Genomics. 2009;93(2):105-111. 31. Biankin AV, Waddell N, Kassahn KS, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012;491(7424):399-405. 32. Bonifacino JS, Dasso M, Harford JB, et al. Current Protocols in Cell Biology. New York: John Wiley & Sons; 2003. 33. Nagy A. Manipulating the Mouse Embryo: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2002. 34. Evans M. Discovering pluripotency: 30 years of mouse embry-onic stem cells. Nat Rev Mol Cell Biol. 2011;12(10):680-686. 35. Steitz JA. In: Hannon GH, ed. RNAi: A Guide to Gene Silenc-ing. New York: Cold Spring Harbor Laboratory Press; 2003.Brunicardi_Ch15_p0479-p0510.indd 50818/02/19 11:12 AM 509MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15 36. Rao DD, Senzer N, Wang Z, Kumar P, Jay CM, Nemunaitis J. Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application. Methods
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Surgery_Schwartz. of sequencing technologies. Genomics. 2009;93(2):105-111. 31. Biankin AV, Waddell N, Kassahn KS, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012;491(7424):399-405. 32. Bonifacino JS, Dasso M, Harford JB, et al. Current Protocols in Cell Biology. New York: John Wiley & Sons; 2003. 33. Nagy A. Manipulating the Mouse Embryo: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2002. 34. Evans M. Discovering pluripotency: 30 years of mouse embry-onic stem cells. Nat Rev Mol Cell Biol. 2011;12(10):680-686. 35. Steitz JA. In: Hannon GH, ed. RNAi: A Guide to Gene Silenc-ing. New York: Cold Spring Harbor Laboratory Press; 2003.Brunicardi_Ch15_p0479-p0510.indd 50818/02/19 11:12 AM 509MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15 36. Rao DD, Senzer N, Wang Z, Kumar P, Jay CM, Nemunaitis J. Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application. Methods
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OF DISEASE, AND PRECISION SURGERYCHAPTER 15 36. Rao DD, Senzer N, Wang Z, Kumar P, Jay CM, Nemunaitis J. Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application. Methods Mol Biol. 2013; 942:259-278. 37. Rao DD, Maples PB, Senzer N, et al. Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference. Cancer Gene Ther. 2010;17(11):780-791. 38. MacRae IJ, Zhou K, Li F, et al. Structural basis for double-stranded RNA processing by dicer. Science. 2006;311(5758): 195-198. 39. Senzer N, Rao D, Nemunaitis J. Letter to the editor: does dicer expression affect shRNA processing? Gene Regul Syst Bio. 2009;3:103-104. 40. Senzer N, Barve M, Kuhn J, et al. Phase I trial of “bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell” vac-cine (FANG) in advanced cancer. Mol Ther. 2012;20(3):679-686. 41. Liu SH, Patel S, Gingras MC, et al. PDX-1: demonstra-tion of oncogenic properties in pancreatic cancer. Cancer. 2011;117(4):723-733. 42. Templeton
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Surgery_Schwartz. OF DISEASE, AND PRECISION SURGERYCHAPTER 15 36. Rao DD, Senzer N, Wang Z, Kumar P, Jay CM, Nemunaitis J. Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application. Methods Mol Biol. 2013; 942:259-278. 37. Rao DD, Maples PB, Senzer N, et al. Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference. Cancer Gene Ther. 2010;17(11):780-791. 38. MacRae IJ, Zhou K, Li F, et al. Structural basis for double-stranded RNA processing by dicer. Science. 2006;311(5758): 195-198. 39. Senzer N, Rao D, Nemunaitis J. Letter to the editor: does dicer expression affect shRNA processing? Gene Regul Syst Bio. 2009;3:103-104. 40. Senzer N, Barve M, Kuhn J, et al. Phase I trial of “bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell” vac-cine (FANG) in advanced cancer. Mol Ther. 2012;20(3):679-686. 41. Liu SH, Patel S, Gingras MC, et al. PDX-1: demonstra-tion of oncogenic properties in pancreatic cancer. Cancer. 2011;117(4):723-733. 42. Templeton
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advanced cancer. Mol Ther. 2012;20(3):679-686. 41. Liu SH, Patel S, Gingras MC, et al. PDX-1: demonstra-tion of oncogenic properties in pancreatic cancer. Cancer. 2011;117(4):723-733. 42. Templeton NS, Lasic DD, Frederik PM, et al. Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol. 1997;15(7):647-652. 43. Nemunaitis J, Rao DD, Liu SH, Brunicardi FC. Personalized cancer approach: using RNA interference technology. World J Surg. 2011;35(8):1700-1714. 44. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826. 45. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823. 46. Ran FA, Hsu PD, Wright J, Argarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. 47. Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev
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Surgery_Schwartz. advanced cancer. Mol Ther. 2012;20(3):679-686. 41. Liu SH, Patel S, Gingras MC, et al. PDX-1: demonstra-tion of oncogenic properties in pancreatic cancer. Cancer. 2011;117(4):723-733. 42. Templeton NS, Lasic DD, Frederik PM, et al. Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol. 1997;15(7):647-652. 43. Nemunaitis J, Rao DD, Liu SH, Brunicardi FC. Personalized cancer approach: using RNA interference technology. World J Surg. 2011;35(8):1700-1714. 44. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826. 45. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823. 46. Ran FA, Hsu PD, Wright J, Argarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. 47. Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev
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F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. 47. Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol. 2010;64:475-493. 48. Ma Y, Zhang L, Huang X. Genome modification by CRISPR/Cas9. FEBS J. 2014;281(23):5186-5193. 49. Lu XJ, Xue HY, Ke ZP, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet. 2015;52(5):289-296. 50. Saleh-Gohari N, Helleday T. Conservative homologous recom-bination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 2004;32(12):3683-3688. 51. Farboud B. Targeted genome editing in Caenorhabditis elegans using CRISPR/Cas9. Wiley Interdiscip Rev Dev Biol. 2017;6(6). 52. Liu J, Zhou Y, Qi X, et al. CRISPR/Cas9 in zebrafish: an effi-cient combination for human genetic diseases modeling. Hum Genet. 2017;136(1):1-12. 53. Ren X, Holsteens K, Li H, et al. Genome editing in
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Surgery_Schwartz. F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. 47. Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol. 2010;64:475-493. 48. Ma Y, Zhang L, Huang X. Genome modification by CRISPR/Cas9. FEBS J. 2014;281(23):5186-5193. 49. Lu XJ, Xue HY, Ke ZP, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet. 2015;52(5):289-296. 50. Saleh-Gohari N, Helleday T. Conservative homologous recom-bination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 2004;32(12):3683-3688. 51. Farboud B. Targeted genome editing in Caenorhabditis elegans using CRISPR/Cas9. Wiley Interdiscip Rev Dev Biol. 2017;6(6). 52. Liu J, Zhou Y, Qi X, et al. CRISPR/Cas9 in zebrafish: an effi-cient combination for human genetic diseases modeling. Hum Genet. 2017;136(1):1-12. 53. Ren X, Holsteens K, Li H, et al. Genome editing in
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J, Zhou Y, Qi X, et al. CRISPR/Cas9 in zebrafish: an effi-cient combination for human genetic diseases modeling. Hum Genet. 2017;136(1):1-12. 53. Ren X, Holsteens K, Li H, et al. Genome editing in Dro-sophila melanogaster: from basic genome engineering to the multipurpose CRISPR-Cas9 system. Sci China Life Sci. 2017;60(5):476-489. 54. Chadwick AC, Musunuru K. Treatment of dyslipidemia using CRISPR/Cas9 genome editing. Curr Atheroscler Rep. 2017;19(7):32. 55. Zou Q, Wang X, Liu Y, et al. Generation of gene-target dogs using CRISPR/Cas9 system. J Mol Cell Biol. 2015;7(6):580-583. 56. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cyno-molgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4):836-843. 57. Krishan K, Kanchan T, Singh B. Human genome editing and ethical considerations. Sci Eng Ethics. 2016;22(2):597-599. 58. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;
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Surgery_Schwartz. J, Zhou Y, Qi X, et al. CRISPR/Cas9 in zebrafish: an effi-cient combination for human genetic diseases modeling. Hum Genet. 2017;136(1):1-12. 53. Ren X, Holsteens K, Li H, et al. Genome editing in Dro-sophila melanogaster: from basic genome engineering to the multipurpose CRISPR-Cas9 system. Sci China Life Sci. 2017;60(5):476-489. 54. Chadwick AC, Musunuru K. Treatment of dyslipidemia using CRISPR/Cas9 genome editing. Curr Atheroscler Rep. 2017;19(7):32. 55. Zou Q, Wang X, Liu Y, et al. Generation of gene-target dogs using CRISPR/Cas9 system. J Mol Cell Biol. 2015;7(6):580-583. 56. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cyno-molgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4):836-843. 57. Krishan K, Kanchan T, Singh B. Human genome editing and ethical considerations. Sci Eng Ethics. 2016;22(2):597-599. 58. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;
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genome editing and ethical considerations. Sci Eng Ethics. 2016;22(2):597-599. 58. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017; 548(7668):413-419. 59. Ledford H. CRISPR fixes disease gene in viable human embryos. Nature. 2017;548(7665):13-14. 60. Ormond KE, Mortlock DP, Scholes DT, et al. Human germline genome editing. Am J Hum Genet. 2017;101(2):167-176.Brunicardi_Ch15_p0479-p0510.indd 50918/02/19 11:12 AM
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Surgery_Schwartz. genome editing and ethical considerations. Sci Eng Ethics. 2016;22(2):597-599. 58. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017; 548(7668):413-419. 59. Ledford H. CRISPR fixes disease gene in viable human embryos. Nature. 2017;548(7665):13-14. 60. Ormond KE, Mortlock DP, Scholes DT, et al. Human germline genome editing. Am J Hum Genet. 2017;101(2):167-176.Brunicardi_Ch15_p0479-p0510.indd 50918/02/19 11:12 AM
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The Skin and Subcutaneous TissuePatrick Harbour and David H. Song 16chapterINTRODUCTIONThe skin is a complex organ encompassing the body’s surface and is continuous with the mucous membranes. Accounting for approximately 15% of total body weight, it is the largest organ in the human body. Enabled by an array of tissue and cell types, intact skin protects the body from external insults. However, the skin is also the source of a myriad of pathologies that include inflammatory disorders, mechanical and thermal injuries, infec-tious diseases, and benign and malignant tumors. The intrica-cies and complexities of this organ and associated pathologies are reasons the skin and subcutaneous tissue remain of great interest and require the attention of various surgical disciplines that include plastic surgery, dermatology, general surgery, and surgical oncology.ANATOMY AND HISTOLOGYBackgroundIt is important that surgeons understand completely the cutane-ous anatomy and its variability as they
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Surgery_Schwartz. The Skin and Subcutaneous TissuePatrick Harbour and David H. Song 16chapterINTRODUCTIONThe skin is a complex organ encompassing the body’s surface and is continuous with the mucous membranes. Accounting for approximately 15% of total body weight, it is the largest organ in the human body. Enabled by an array of tissue and cell types, intact skin protects the body from external insults. However, the skin is also the source of a myriad of pathologies that include inflammatory disorders, mechanical and thermal injuries, infec-tious diseases, and benign and malignant tumors. The intrica-cies and complexities of this organ and associated pathologies are reasons the skin and subcutaneous tissue remain of great interest and require the attention of various surgical disciplines that include plastic surgery, dermatology, general surgery, and surgical oncology.ANATOMY AND HISTOLOGYBackgroundIt is important that surgeons understand completely the cutane-ous anatomy and its variability as they
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surgery, dermatology, general surgery, and surgical oncology.ANATOMY AND HISTOLOGYBackgroundIt is important that surgeons understand completely the cutane-ous anatomy and its variability as they play an enormous role in patient health and satisfaction. The skin is made up of tissues derived from both the ectodermal and mesodermal germ cell layers.1 Three distinct tissue layers comprise the organ, and differ in composition based on location, age, sex, and ethnicity, among other variables. The outermost layer is the epidermis, which is predominantly characterized by a protective, highly keratinized layer of cells. The next layer is the dermis, which is made up of an organized collagen network to support the numerous epider-mal appendages, neurovascular structures, and supportive cells within the skin. The fatty layer below the dermis is collectively known as the hypodermis and functions in body processes of thermoregulation and energy storage, among others. These three distinct layers
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Surgery_Schwartz. surgery, dermatology, general surgery, and surgical oncology.ANATOMY AND HISTOLOGYBackgroundIt is important that surgeons understand completely the cutane-ous anatomy and its variability as they play an enormous role in patient health and satisfaction. The skin is made up of tissues derived from both the ectodermal and mesodermal germ cell layers.1 Three distinct tissue layers comprise the organ, and differ in composition based on location, age, sex, and ethnicity, among other variables. The outermost layer is the epidermis, which is predominantly characterized by a protective, highly keratinized layer of cells. The next layer is the dermis, which is made up of an organized collagen network to support the numerous epider-mal appendages, neurovascular structures, and supportive cells within the skin. The fatty layer below the dermis is collectively known as the hypodermis and functions in body processes of thermoregulation and energy storage, among others. These three distinct layers
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the skin. The fatty layer below the dermis is collectively known as the hypodermis and functions in body processes of thermoregulation and energy storage, among others. These three distinct layers function together harmoniously and participate in numerous activities essential to life.2EpidermisThe epidermis is the outermost layer of the cutaneous tissue, and consists primarily of continually regenerating keratinocytes. The tissue is also stratified, forming four to five histologically distinct layers, depending on the location in the body. These layers are, from deep to superficial, the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum (Fig. 16-1). The different layers of the epidermis represent layers of keratinocytes at differing stages of their approximately thirty-day life cycle. A minority of other cell types are found in different layers of the epidermis as well. Some of these cells are permanent residents, while others are visitors from
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Surgery_Schwartz. the skin. The fatty layer below the dermis is collectively known as the hypodermis and functions in body processes of thermoregulation and energy storage, among others. These three distinct layers function together harmoniously and participate in numerous activities essential to life.2EpidermisThe epidermis is the outermost layer of the cutaneous tissue, and consists primarily of continually regenerating keratinocytes. The tissue is also stratified, forming four to five histologically distinct layers, depending on the location in the body. These layers are, from deep to superficial, the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum (Fig. 16-1). The different layers of the epidermis represent layers of keratinocytes at differing stages of their approximately thirty-day life cycle. A minority of other cell types are found in different layers of the epidermis as well. Some of these cells are permanent residents, while others are visitors from
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approximately thirty-day life cycle. A minority of other cell types are found in different layers of the epidermis as well. Some of these cells are permanent residents, while others are visitors from other parts of the body. All the epidermal appendages, such as sweat glands and pilosebaceous follicles, are derived from this tissue. The thickness of the epidermis is quite variable with regard to location and age, ranging from 75 to 150 µm in thin skin (eyelids) to 0.4 to 1.5 mm in thick skin (palms and soles).2 The epidermis lacks any vascular Introduction513Anatomy and Histology513Background / 513Epidermis / 513Epidermal Components / 514Epidermal Appendages / 515Dermal Components / 516Cells / 516Cutaneous Vasculature / 516Cutaneous Innervation / 517Hypodermis / 517Inflammatory Conditions517Hidradenitis Suppurativa / 517Pyoderma Gangrenosum / 517Epidermal Necrolysis / 517Injuries518Radiation-Induced Injuries / 518Trauma-Induced Injuries / 519Caustic Injury / 520Thermal Injury /
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Surgery_Schwartz. approximately thirty-day life cycle. A minority of other cell types are found in different layers of the epidermis as well. Some of these cells are permanent residents, while others are visitors from other parts of the body. All the epidermal appendages, such as sweat glands and pilosebaceous follicles, are derived from this tissue. The thickness of the epidermis is quite variable with regard to location and age, ranging from 75 to 150 µm in thin skin (eyelids) to 0.4 to 1.5 mm in thick skin (palms and soles).2 The epidermis lacks any vascular Introduction513Anatomy and Histology513Background / 513Epidermis / 513Epidermal Components / 514Epidermal Appendages / 515Dermal Components / 516Cells / 516Cutaneous Vasculature / 516Cutaneous Innervation / 517Hypodermis / 517Inflammatory Conditions517Hidradenitis Suppurativa / 517Pyoderma Gangrenosum / 517Epidermal Necrolysis / 517Injuries518Radiation-Induced Injuries / 518Trauma-Induced Injuries / 519Caustic Injury / 520Thermal Injury /
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Suppurativa / 517Pyoderma Gangrenosum / 517Epidermal Necrolysis / 517Injuries518Radiation-Induced Injuries / 518Trauma-Induced Injuries / 519Caustic Injury / 520Thermal Injury / 521Pressure Injury / 523Bioengineered Skin Substitutes524Bacterial Infections of the Skin and Subcutaneous Tissue524Introduction / 524Uncomplicated Skin Infections / 524Complicated Skin Infections / 524Actinomycosis / 526Viral Infections with Surgical Implications526Human Papillomavirus Infections / 526Cutaneous Manifestations of Human Immunodeficiency Virus / 527Benign Tumors527Hemangioma / 527Nevi / 527Cystic Lesions / 527Keratosis / 528Soft Tissue Tumors / 528Neural Tumors / 528Malignant Tumors528Basal Cell Carcinoma / 528Squamous Cell Carcinoma / 529Melanoma / 530Merkel Cell Carcinoma / 534Kaposi’s Sarcoma / 535Dermatofibrosarcoma Protuberans / 535Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma) / 535Angiosarcoma / 535Extramammary Paget’s Disease /
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Surgery_Schwartz. Suppurativa / 517Pyoderma Gangrenosum / 517Epidermal Necrolysis / 517Injuries518Radiation-Induced Injuries / 518Trauma-Induced Injuries / 519Caustic Injury / 520Thermal Injury / 521Pressure Injury / 523Bioengineered Skin Substitutes524Bacterial Infections of the Skin and Subcutaneous Tissue524Introduction / 524Uncomplicated Skin Infections / 524Complicated Skin Infections / 524Actinomycosis / 526Viral Infections with Surgical Implications526Human Papillomavirus Infections / 526Cutaneous Manifestations of Human Immunodeficiency Virus / 527Benign Tumors527Hemangioma / 527Nevi / 527Cystic Lesions / 527Keratosis / 528Soft Tissue Tumors / 528Neural Tumors / 528Malignant Tumors528Basal Cell Carcinoma / 528Squamous Cell Carcinoma / 529Melanoma / 530Merkel Cell Carcinoma / 534Kaposi’s Sarcoma / 535Dermatofibrosarcoma Protuberans / 535Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma) / 535Angiosarcoma / 535Extramammary Paget’s Disease /
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Surgery_Schwartz_3487
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Sarcoma / 535Dermatofibrosarcoma Protuberans / 535Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma) / 535Angiosarcoma / 535Extramammary Paget’s Disease / 536Conclusion536Brunicardi_Ch16_p0511-p0540.indd 51319/02/19 3:08 PM 514Hair shaftStratum corneumPigment ligamentStratum germinativumStratum spinosumStratum basaleArrector pili muscleSebaceous glandHair folliclePapilla of hairBlood andlymph vesselsNerve ÿberSweatporeDermalpapillaSensory nerve ending for touchEpidermisDermisSubcutis(hypodermis)VeinArteryPaciniancorpuscleSweatglandFigure 16-1. Schematic representation of the skin and its appendages. Note that the root of the hair follicle may extend beneath the dermis into the subcutis.structures and obtains all nutrients from the dermal vasculature by diffusion.3Epidermal ComponentsKeratinocytes. Keratinocytes typically make up about 90% of the cells of the epidermis. These cells have four to five distinct stages in their life cycle, each
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Surgery_Schwartz. Sarcoma / 535Dermatofibrosarcoma Protuberans / 535Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma) / 535Angiosarcoma / 535Extramammary Paget’s Disease / 536Conclusion536Brunicardi_Ch16_p0511-p0540.indd 51319/02/19 3:08 PM 514Hair shaftStratum corneumPigment ligamentStratum germinativumStratum spinosumStratum basaleArrector pili muscleSebaceous glandHair folliclePapilla of hairBlood andlymph vesselsNerve ÿberSweatporeDermalpapillaSensory nerve ending for touchEpidermisDermisSubcutis(hypodermis)VeinArteryPaciniancorpuscleSweatglandFigure 16-1. Schematic representation of the skin and its appendages. Note that the root of the hair follicle may extend beneath the dermis into the subcutis.structures and obtains all nutrients from the dermal vasculature by diffusion.3Epidermal ComponentsKeratinocytes. Keratinocytes typically make up about 90% of the cells of the epidermis. These cells have four to five distinct stages in their life cycle, each
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Surgery_Schwartz_3488
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by diffusion.3Epidermal ComponentsKeratinocytes. Keratinocytes typically make up about 90% of the cells of the epidermis. These cells have four to five distinct stages in their life cycle, each visibly different under light microscopy. The stratum basale, or germinative layer, is a deep, single layer of asynchronous, continuously rep-licating cuboidal to columnar epithelial cells and is the 1beginning of the life cycle of the keratinocytes of the epidermis. This layer is bound to its basement membrane by complexes made of keratin filaments and anchoring structures called hemidesmosomes. They are bound to other keratinocytes by structures called desmosomes. High mitotic activity and thus large nuclei and basophilic staining characterize the stratum basale on light microscopy. This layer also lines the epidermal appendages that reside largely within the substance of the der-mis and later serves as a regenerative source of epithelium in the event of partial thickness wounds.Key
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Surgery_Schwartz. by diffusion.3Epidermal ComponentsKeratinocytes. Keratinocytes typically make up about 90% of the cells of the epidermis. These cells have four to five distinct stages in their life cycle, each visibly different under light microscopy. The stratum basale, or germinative layer, is a deep, single layer of asynchronous, continuously rep-licating cuboidal to columnar epithelial cells and is the 1beginning of the life cycle of the keratinocytes of the epidermis. This layer is bound to its basement membrane by complexes made of keratin filaments and anchoring structures called hemidesmosomes. They are bound to other keratinocytes by structures called desmosomes. High mitotic activity and thus large nuclei and basophilic staining characterize the stratum basale on light microscopy. This layer also lines the epidermal appendages that reside largely within the substance of the der-mis and later serves as a regenerative source of epithelium in the event of partial thickness wounds.Key
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Surgery_Schwartz_3489
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layer also lines the epidermal appendages that reside largely within the substance of the der-mis and later serves as a regenerative source of epithelium in the event of partial thickness wounds.Key Points1 The epidermis consists of continually regenerating strati-fied epithelium, and 90% of cells are ectodermally derived keratinocytes.2 Pilosebaceous units are lined by the germinal epithelium of the epidermis and thus serve as an important source of epidermal regeneration after partial-thickness injury or split-thickness skin graft.3 Dermal fibers are predominantly made of type I and III collagen in a 4:1 ratio. They are responsible for the mechanical resistance of skin.4 The drugs most commonly associated with epidermal necrolysis include aromatic anticonvulsants, sulfonamides, allopurinol, oxicams (nonsteroidal anti-inflammatory drugs), and nevirapine.5 In wounds being allowed to heal secondarily, negative pressure wound therapy can increase the rate of granula-tion tissue
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Surgery_Schwartz. layer also lines the epidermal appendages that reside largely within the substance of the der-mis and later serves as a regenerative source of epithelium in the event of partial thickness wounds.Key Points1 The epidermis consists of continually regenerating strati-fied epithelium, and 90% of cells are ectodermally derived keratinocytes.2 Pilosebaceous units are lined by the germinal epithelium of the epidermis and thus serve as an important source of epidermal regeneration after partial-thickness injury or split-thickness skin graft.3 Dermal fibers are predominantly made of type I and III collagen in a 4:1 ratio. They are responsible for the mechanical resistance of skin.4 The drugs most commonly associated with epidermal necrolysis include aromatic anticonvulsants, sulfonamides, allopurinol, oxicams (nonsteroidal anti-inflammatory drugs), and nevirapine.5 In wounds being allowed to heal secondarily, negative pressure wound therapy can increase the rate of granula-tion tissue
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Surgery_Schwartz_3490
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Surgery_Schwartz
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allopurinol, oxicams (nonsteroidal anti-inflammatory drugs), and nevirapine.5 In wounds being allowed to heal secondarily, negative pressure wound therapy can increase the rate of granula-tion tissue formation.6 Staphylococcus aureus is the most common isolate of all skin infections. Impetigo, cellulitis, erysipelas, folliculitis, furuncles, and simple abscesses are examples of uncompli-cated infections, whereas deep-tissue infections, extensive cellulitis, necrotizing fasciitis, and myonecrosis are exam-ples of complicated infections.7 Hemangiomas arise from benign proliferation of endothe-lial cells surrounding blood-filled cavities. They most commonly present after birth, rapidly grow during the first year of life, and gradually involute in most cases.8 Basal cell carcinoma represents the most common tumor diagnosed in the United States, and the nodular variant is the most common subtype. The natural progression of basal cell carcinoma is one of local invasion rather than distant
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Surgery_Schwartz. allopurinol, oxicams (nonsteroidal anti-inflammatory drugs), and nevirapine.5 In wounds being allowed to heal secondarily, negative pressure wound therapy can increase the rate of granula-tion tissue formation.6 Staphylococcus aureus is the most common isolate of all skin infections. Impetigo, cellulitis, erysipelas, folliculitis, furuncles, and simple abscesses are examples of uncompli-cated infections, whereas deep-tissue infections, extensive cellulitis, necrotizing fasciitis, and myonecrosis are exam-ples of complicated infections.7 Hemangiomas arise from benign proliferation of endothe-lial cells surrounding blood-filled cavities. They most commonly present after birth, rapidly grow during the first year of life, and gradually involute in most cases.8 Basal cell carcinoma represents the most common tumor diagnosed in the United States, and the nodular variant is the most common subtype. The natural progression of basal cell carcinoma is one of local invasion rather than distant
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Surgery_Schwartz_3491
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the most common tumor diagnosed in the United States, and the nodular variant is the most common subtype. The natural progression of basal cell carcinoma is one of local invasion rather than distant metastasis.9 Squamous cell carcinoma is the second most common skin cancer, and typically arises from an actinic keratosis precur-sor. Primary treatment modalities are surgical excision and Mohs microsurgery. Cautery and ablation, cryotherapy, drug therapy, and radiation therapy are alternative treatments.10 Tumor thickness, ulceration, and mitotic rate are the most important prognostic indicators of survival in melanoma. Sentinel lymph node biopsy is often used to stage indi-viduals with biopsy-proven high risk melanoma and clini-cally node-negative disease.Brunicardi_Ch16_p0511-p0540.indd 51419/02/19 3:08 PM 515THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16The next layer is the stratum spinosum, or “spiny” layer. This layer is from five to fifteen cells in thickness and is so named due
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Surgery_Schwartz. the most common tumor diagnosed in the United States, and the nodular variant is the most common subtype. The natural progression of basal cell carcinoma is one of local invasion rather than distant metastasis.9 Squamous cell carcinoma is the second most common skin cancer, and typically arises from an actinic keratosis precur-sor. Primary treatment modalities are surgical excision and Mohs microsurgery. Cautery and ablation, cryotherapy, drug therapy, and radiation therapy are alternative treatments.10 Tumor thickness, ulceration, and mitotic rate are the most important prognostic indicators of survival in melanoma. Sentinel lymph node biopsy is often used to stage indi-viduals with biopsy-proven high risk melanoma and clini-cally node-negative disease.Brunicardi_Ch16_p0511-p0540.indd 51419/02/19 3:08 PM 515THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16The next layer is the stratum spinosum, or “spiny” layer. This layer is from five to fifteen cells in thickness and is so named due
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Surgery_Schwartz_3492
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51419/02/19 3:08 PM 515THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16The next layer is the stratum spinosum, or “spiny” layer. This layer is from five to fifteen cells in thickness and is so named due to the spinous appearance of the intercellular des-mosomal attachments under light microscopy. The production of keratin in this cell layer is responsible for their eosinophilic appearance on hematoxylin and eosin (H&E) staining.As the keratinocytes continue to migrate superficially, they begin to flatten and develop basophilic keratohyalin gran-ules. There are also structures called lamellar granules within these cells that contain the lipids and glycolipids that will ulti-mately undergo exocytosis to produce the lipid layer around the cells. It is in this layer that the keratinocytes manufacture many of the structures that will eventually serve to protect the skin and underlying tissues from environmental insult.4 At the super-ficial aspect of this layer, the keratinocytes begin to
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Surgery_Schwartz. 51419/02/19 3:08 PM 515THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16The next layer is the stratum spinosum, or “spiny” layer. This layer is from five to fifteen cells in thickness and is so named due to the spinous appearance of the intercellular des-mosomal attachments under light microscopy. The production of keratin in this cell layer is responsible for their eosinophilic appearance on hematoxylin and eosin (H&E) staining.As the keratinocytes continue to migrate superficially, they begin to flatten and develop basophilic keratohyalin gran-ules. There are also structures called lamellar granules within these cells that contain the lipids and glycolipids that will ulti-mately undergo exocytosis to produce the lipid layer around the cells. It is in this layer that the keratinocytes manufacture many of the structures that will eventually serve to protect the skin and underlying tissues from environmental insult.4 At the super-ficial aspect of this layer, the keratinocytes begin to
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Surgery_Schwartz_3493
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Surgery_Schwartz
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many of the structures that will eventually serve to protect the skin and underlying tissues from environmental insult.4 At the super-ficial aspect of this layer, the keratinocytes begin to undergo programmed cell death, losing all cellular structures except for the keratin filaments and their associated proteins. In thick skin, such as that found on the palms and soles, there is a layer of flat, translucent keratinocytes called the stratum lucidum.The final stage of the keratinocyte life cycle results in the layer of the epidermis known as the stratum corneum, or cor-nified layer. The protein-rich, flattened keratinocytes are now anucleate and surrounded by a lipid-rich matrix. Together the cells and surrounding matrix of this layer serve to protect the tissue from mechanical, chemical, and bacterial disruption while preventing insensible water losses through the skin.4,5Langerhans Cells. Of the cells in the epidermis, 3% to 6% are immune cells known as Langerhans cells.6 Typically
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Surgery_Schwartz. many of the structures that will eventually serve to protect the skin and underlying tissues from environmental insult.4 At the super-ficial aspect of this layer, the keratinocytes begin to undergo programmed cell death, losing all cellular structures except for the keratin filaments and their associated proteins. In thick skin, such as that found on the palms and soles, there is a layer of flat, translucent keratinocytes called the stratum lucidum.The final stage of the keratinocyte life cycle results in the layer of the epidermis known as the stratum corneum, or cor-nified layer. The protein-rich, flattened keratinocytes are now anucleate and surrounded by a lipid-rich matrix. Together the cells and surrounding matrix of this layer serve to protect the tissue from mechanical, chemical, and bacterial disruption while preventing insensible water losses through the skin.4,5Langerhans Cells. Of the cells in the epidermis, 3% to 6% are immune cells known as Langerhans cells.6 Typically
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Surgery_Schwartz_3494
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and bacterial disruption while preventing insensible water losses through the skin.4,5Langerhans Cells. Of the cells in the epidermis, 3% to 6% are immune cells known as Langerhans cells.6 Typically found within the stratum spinosum, these mobile, dendritic cells inter-digitate between keratinocytes of the epidermis to create a dense network, sampling any antigens that attempt to pass through the cutaneous tissue. Through use of their characteristic rodor racket-shaped Birbeck granules, they take up antigens for pre-sentation to T-cells.7 These monocyte-derived cells represent a large part of the skin’s adaptive immunity. Because of the effec-tiveness of their antigen presentation, Langerhans cells could be utilized as vaccine vehicles in the future.8 The Langerhans cells are functionally impaired by UV radiation, specifically UVB radiation, and may play a role in the development of cutaneous malignancies after UV radiation exposure.9Melanocytes. Within the stratum basale are
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Surgery_Schwartz. and bacterial disruption while preventing insensible water losses through the skin.4,5Langerhans Cells. Of the cells in the epidermis, 3% to 6% are immune cells known as Langerhans cells.6 Typically found within the stratum spinosum, these mobile, dendritic cells inter-digitate between keratinocytes of the epidermis to create a dense network, sampling any antigens that attempt to pass through the cutaneous tissue. Through use of their characteristic rodor racket-shaped Birbeck granules, they take up antigens for pre-sentation to T-cells.7 These monocyte-derived cells represent a large part of the skin’s adaptive immunity. Because of the effec-tiveness of their antigen presentation, Langerhans cells could be utilized as vaccine vehicles in the future.8 The Langerhans cells are functionally impaired by UV radiation, specifically UVB radiation, and may play a role in the development of cutaneous malignancies after UV radiation exposure.9Melanocytes. Within the stratum basale are
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Surgery_Schwartz_3495
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impaired by UV radiation, specifically UVB radiation, and may play a role in the development of cutaneous malignancies after UV radiation exposure.9Melanocytes. Within the stratum basale are melanocytes, the cells responsible for production of the pigment melanin in the skin. These neural crest-derived cells are present in a density of four to ten keratinocytes per melanocytes, and about 500 to 2000 melanocytes per mm2 of cutaneous tissue. This density varies based on location in the body, but differences in skin pig-mentation are based on the activity of individual melanocytes and not the number of melanocytes. In darker-skinned ethnici-ties, melanocytes create and store melanosomes in keratinocytes at a higher rate, but still have a pale-staining cytoplasm on light microscopy. Hemidesmosomes also attach these cells to the basement membrane, but the intercellular desmosomal connec-tions are not present. The melanocytes interact with keratino-cytes of the stratum basale and spinosum
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Surgery_Schwartz. impaired by UV radiation, specifically UVB radiation, and may play a role in the development of cutaneous malignancies after UV radiation exposure.9Melanocytes. Within the stratum basale are melanocytes, the cells responsible for production of the pigment melanin in the skin. These neural crest-derived cells are present in a density of four to ten keratinocytes per melanocytes, and about 500 to 2000 melanocytes per mm2 of cutaneous tissue. This density varies based on location in the body, but differences in skin pig-mentation are based on the activity of individual melanocytes and not the number of melanocytes. In darker-skinned ethnici-ties, melanocytes create and store melanosomes in keratinocytes at a higher rate, but still have a pale-staining cytoplasm on light microscopy. Hemidesmosomes also attach these cells to the basement membrane, but the intercellular desmosomal connec-tions are not present. The melanocytes interact with keratino-cytes of the stratum basale and spinosum
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Surgery_Schwartz_3496
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also attach these cells to the basement membrane, but the intercellular desmosomal connec-tions are not present. The melanocytes interact with keratino-cytes of the stratum basale and spinosum via long cytoplasmic extensions leading to invaginations in several keratinocytes. Tyrosinase is created and distributed into melanosomes, and these organelles travel along the dendritic processes to eventu-ally become phagocytized by keratinocytes and distributed in a supranuclear orientation. This umbrella-like cap then serves to protect the nuclear material from damage by radiation; this could explain why light-skinned ethnicities are more prone to the development of cutaneous malignancies.10,11 Melanocytes express the bcl-2 protein, S100 protein, and vimentin, which are important in the pathology and histologic diagnosis of disorders of melanocytes.Merkel Cells. Merkel cells are slow-adapting mechanorecep-tors of unclear origin essential for light touch sensation. Thus, they typically
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Surgery_Schwartz. also attach these cells to the basement membrane, but the intercellular desmosomal connec-tions are not present. The melanocytes interact with keratino-cytes of the stratum basale and spinosum via long cytoplasmic extensions leading to invaginations in several keratinocytes. Tyrosinase is created and distributed into melanosomes, and these organelles travel along the dendritic processes to eventu-ally become phagocytized by keratinocytes and distributed in a supranuclear orientation. This umbrella-like cap then serves to protect the nuclear material from damage by radiation; this could explain why light-skinned ethnicities are more prone to the development of cutaneous malignancies.10,11 Melanocytes express the bcl-2 protein, S100 protein, and vimentin, which are important in the pathology and histologic diagnosis of disorders of melanocytes.Merkel Cells. Merkel cells are slow-adapting mechanorecep-tors of unclear origin essential for light touch sensation. Thus, they typically
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Surgery_Schwartz_3497
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Surgery_Schwartz
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and histologic diagnosis of disorders of melanocytes.Merkel Cells. Merkel cells are slow-adapting mechanorecep-tors of unclear origin essential for light touch sensation. Thus, they typically aggregate among basal keratinocytes of the skin in areas where light tactile sensation is warranted, such as the digits, lips, and bases of some hair follicles.12-14 They are joined to keratinocytes in the basal layer by desmosomes and have dense neurosecretory granules containing peptides. These neu-rosecretory granules allow communication with the CNS via afferent, unmyelinated nerve fibers that contact the basolateral portion of the cell via expanded terminal discs.3 The clinical significance of Merkel cells arises in the setting of Merkel cell carcinoma, a rare, but difficult-to-treat malignancy.Lymphocytes. Less than 1% of the cells in the epidermis are lymphocytes, and these are found primarily within the basal layer of keratinocytes. They typically express an effector memory T-cell
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Surgery_Schwartz. and histologic diagnosis of disorders of melanocytes.Merkel Cells. Merkel cells are slow-adapting mechanorecep-tors of unclear origin essential for light touch sensation. Thus, they typically aggregate among basal keratinocytes of the skin in areas where light tactile sensation is warranted, such as the digits, lips, and bases of some hair follicles.12-14 They are joined to keratinocytes in the basal layer by desmosomes and have dense neurosecretory granules containing peptides. These neu-rosecretory granules allow communication with the CNS via afferent, unmyelinated nerve fibers that contact the basolateral portion of the cell via expanded terminal discs.3 The clinical significance of Merkel cells arises in the setting of Merkel cell carcinoma, a rare, but difficult-to-treat malignancy.Lymphocytes. Less than 1% of the cells in the epidermis are lymphocytes, and these are found primarily within the basal layer of keratinocytes. They typically express an effector memory T-cell
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Surgery_Schwartz_3498
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Surgery_Schwartz
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than 1% of the cells in the epidermis are lymphocytes, and these are found primarily within the basal layer of keratinocytes. They typically express an effector memory T-cell phenotype.15,16Toker Cells. Toker cells are found in the epidermis of the nip-ple in 10% of both males and females and were first described in 1970. While distinct from Paget’s cells, immunohistochemical studies have implicated them as a possible source of Paget’s disease of the nipple.17-20Epidermal AppendagesSweat Glands. Sweat glands, like other epidermal appendages, are derived from the embryologic ectoderm, but the bulk of their substance resides within the dermis. Their structure consists of a tubular-shaped exocrine gland and excretory duct. Eccrine sweat glands make up a majority of the sweat glands in the body and are extremely important to the process of thermoregu-lation. Solutes are released into the gland via exocytosis. They are present in greatest numbers on the palms, soles, axillae, and forehead.
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Surgery_Schwartz. than 1% of the cells in the epidermis are lymphocytes, and these are found primarily within the basal layer of keratinocytes. They typically express an effector memory T-cell phenotype.15,16Toker Cells. Toker cells are found in the epidermis of the nip-ple in 10% of both males and females and were first described in 1970. While distinct from Paget’s cells, immunohistochemical studies have implicated them as a possible source of Paget’s disease of the nipple.17-20Epidermal AppendagesSweat Glands. Sweat glands, like other epidermal appendages, are derived from the embryologic ectoderm, but the bulk of their substance resides within the dermis. Their structure consists of a tubular-shaped exocrine gland and excretory duct. Eccrine sweat glands make up a majority of the sweat glands in the body and are extremely important to the process of thermoregu-lation. Solutes are released into the gland via exocytosis. They are present in greatest numbers on the palms, soles, axillae, and forehead.
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Surgery_Schwartz_3499
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and are extremely important to the process of thermoregu-lation. Solutes are released into the gland via exocytosis. They are present in greatest numbers on the palms, soles, axillae, and forehead. Collectively they produce approximately 10 L/d in an adult. These glands are the most effective means of temperature regulation in humans via evaporative heat loss.A second type of sweat gland, known as the apocrine sweat gland, is found around the axilla, anus, areola, eyelid, and external auditory canal. The cells in this gland undergo an excretion process that involves decapitation of part of the cell. These apocrine glands are typically activated by sex hormones and thus activate around the time of puberty. The secretion from apocrine glands is initially odorless, but bacteria in the region may cause an odor to develop. Pheromone production may have been a function of the apocrine glands, but this may now be vestigial. While eccrine sweat glands are activated by the cho-linergic system,
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Surgery_Schwartz. and are extremely important to the process of thermoregu-lation. Solutes are released into the gland via exocytosis. They are present in greatest numbers on the palms, soles, axillae, and forehead. Collectively they produce approximately 10 L/d in an adult. These glands are the most effective means of temperature regulation in humans via evaporative heat loss.A second type of sweat gland, known as the apocrine sweat gland, is found around the axilla, anus, areola, eyelid, and external auditory canal. The cells in this gland undergo an excretion process that involves decapitation of part of the cell. These apocrine glands are typically activated by sex hormones and thus activate around the time of puberty. The secretion from apocrine glands is initially odorless, but bacteria in the region may cause an odor to develop. Pheromone production may have been a function of the apocrine glands, but this may now be vestigial. While eccrine sweat glands are activated by the cho-linergic system,
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Surgery_Schwartz_3500
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Surgery_Schwartz
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may cause an odor to develop. Pheromone production may have been a function of the apocrine glands, but this may now be vestigial. While eccrine sweat glands are activated by the cho-linergic system, apocrine glands are activated by the adrenergic system.There is also a third type of sweat gland called apoeccrine. This is similar to an apocrine gland but opens directly to the skin surface and does not present until puberty. 21 Both types of glands are surrounded by a layer of myoepithelial cells that can contract and assist in the excretion of glandular contents to the skin surface.Pilosebaceous Units. A pilosebaceous unit is a multicompo-nent unit made up of a hair follicle, sebaceous gland, an erector pili muscle, and a sensory organ. These units are responsible for the production of hair and sebum and are present almost entirely Brunicardi_Ch16_p0511-p0540.indd 51519/02/19 3:08 PM 516SPECIFIC CONSIDERATIONSPART IIthroughout the body, sparing the palms, soles, and mucosa. They
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Surgery_Schwartz. may cause an odor to develop. Pheromone production may have been a function of the apocrine glands, but this may now be vestigial. While eccrine sweat glands are activated by the cho-linergic system, apocrine glands are activated by the adrenergic system.There is also a third type of sweat gland called apoeccrine. This is similar to an apocrine gland but opens directly to the skin surface and does not present until puberty. 21 Both types of glands are surrounded by a layer of myoepithelial cells that can contract and assist in the excretion of glandular contents to the skin surface.Pilosebaceous Units. A pilosebaceous unit is a multicompo-nent unit made up of a hair follicle, sebaceous gland, an erector pili muscle, and a sensory organ. These units are responsible for the production of hair and sebum and are present almost entirely Brunicardi_Ch16_p0511-p0540.indd 51519/02/19 3:08 PM 516SPECIFIC CONSIDERATIONSPART IIthroughout the body, sparing the palms, soles, and mucosa. They
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Surgery_Schwartz_3501
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Surgery_Schwartz
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and sebum and are present almost entirely Brunicardi_Ch16_p0511-p0540.indd 51519/02/19 3:08 PM 516SPECIFIC CONSIDERATIONSPART IIthroughout the body, sparing the palms, soles, and mucosa. They are lined by the germinal epithelium of the epidermis and thus serve as an important source of epidermal regenera-tion after partial-thickness injury or split-thickness skin graft. The sebaceous glands secrete sebum into the follicle and skin via a duct. The lipid-secreting glands are largely influenced by androgens and become functionally active during puberty. They are present in greatest numbers on the face and scalp.Nails. The nails are keratinaceous structures overlying the dis-tal phalanges of the fingers and toes. The nail is made of three main parts. The proximal portion of the nail, continuous with the germinal nail matrix, is the nail root. The root is an adher-ence point for the nail. The nail plate is the portion of the nail that lies on top of the nail bed, the shape of which is
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Surgery_Schwartz. and sebum and are present almost entirely Brunicardi_Ch16_p0511-p0540.indd 51519/02/19 3:08 PM 516SPECIFIC CONSIDERATIONSPART IIthroughout the body, sparing the palms, soles, and mucosa. They are lined by the germinal epithelium of the epidermis and thus serve as an important source of epidermal regenera-tion after partial-thickness injury or split-thickness skin graft. The sebaceous glands secrete sebum into the follicle and skin via a duct. The lipid-secreting glands are largely influenced by androgens and become functionally active during puberty. They are present in greatest numbers on the face and scalp.Nails. The nails are keratinaceous structures overlying the dis-tal phalanges of the fingers and toes. The nail is made of three main parts. The proximal portion of the nail, continuous with the germinal nail matrix, is the nail root. The root is an adher-ence point for the nail. The nail plate is the portion of the nail that lies on top of the nail bed, the shape of which is
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