Introducing a "How-to" Guide to Embryonic Stem Cell Manipulation
http://www.100md.com
《干细胞学杂志》
Children’s Hospital and Harvard Medical School, Cambridge, MA
Science is frequently advanced by a bold new technology or a critical reagent. Inaccessible experiments suddenly become approachable, and discovery proceeds at an accelerated rate, advancing rapidly until it confronts the next technical barrier or missing link. Embryonic stem cells represent one such broadly enabling technology platform. Their isolation and culture from mouse embryos in 1981 has enabled over 2 decades of inquiry into developmental processes that had previously been largely unexplored. When coupled with methods for homologous recombination and generation of genetically defined mouse strains, embryonic stem cells have provided valuable models of human disease. Ultimately, insights into the mechanisms of cell differentiation and tissue organization made possible by the study of mouse—and more recently, human—embryonic stem cells may one day enable cell replacement therapies. Realizing the promise of regenerative medicine, however, will require meticulous, painstaking attention to the details of embryonic stem cell culture, genetic manipulation, cell differentiation, and cell characterization—and these techniques must be made accessible to all researchers so that these critical reagents can be exploited widely.
With that goal in mind, STEM CELLS is launching a series of methods-based reports penned by leading figures experienced in aspects of embryonic stem cell manipulation. The first in this series, by Tompers and Labosky, provides a step-by-step "how-to" guide to genetic modification of murine embryonic stem cells by electroporation, a critical methodology for all researchers experimenting with embryonic stem cells. Replete with experimental detail, photo-micrographic documentation, and helpful technical pearls, this report walks the practitioner through the steps of cell culture, electroporation, drug selection, picking and expansion of colonies, cryopreservation and thawing of cell lines, DNA isolation for analysis, and preparation of cells for blastocyst injection or morula aggregation. The narrative is instructive without being preachy and will serve the curious experimentalist well.
We encourage and invite future efforts from those of you willing to share your experience and experimental wisdom, including narrative details that might not be found in protocol "handbooks." Manuscripts of considerable interest might include protocols for: A) preparation and characterization of stromal cell lines from various tissues and species that support embryonic and adult/somatic stem cell culture; B) manipulation of retroviral and lentiviral vectors and their expression patterns in mouse and human embryonic stem cells; C) differentiation and characterization of specific tissue lineages from mouse and human embryonic stem cells; D) primary isolation and culture of embryonic stem cells from mouse and other mammalian species; E) nuclear transfer and parthenogenesis, and F) obtaining approval from Institutional Review Boards for work with human embryos and embryonic stem cells. These are but a few of the possible topics that might pique the interest of the mounting wave of scientists eager to learn the latest techniques of embryonic stem cell manipulation. The community of biomedical scientists and the pace of scientific discovery will be well served by demystifying the methodology surrounding embryonic stem cells(George Q. Daley, M.D., Ph)
Science is frequently advanced by a bold new technology or a critical reagent. Inaccessible experiments suddenly become approachable, and discovery proceeds at an accelerated rate, advancing rapidly until it confronts the next technical barrier or missing link. Embryonic stem cells represent one such broadly enabling technology platform. Their isolation and culture from mouse embryos in 1981 has enabled over 2 decades of inquiry into developmental processes that had previously been largely unexplored. When coupled with methods for homologous recombination and generation of genetically defined mouse strains, embryonic stem cells have provided valuable models of human disease. Ultimately, insights into the mechanisms of cell differentiation and tissue organization made possible by the study of mouse—and more recently, human—embryonic stem cells may one day enable cell replacement therapies. Realizing the promise of regenerative medicine, however, will require meticulous, painstaking attention to the details of embryonic stem cell culture, genetic manipulation, cell differentiation, and cell characterization—and these techniques must be made accessible to all researchers so that these critical reagents can be exploited widely.
With that goal in mind, STEM CELLS is launching a series of methods-based reports penned by leading figures experienced in aspects of embryonic stem cell manipulation. The first in this series, by Tompers and Labosky, provides a step-by-step "how-to" guide to genetic modification of murine embryonic stem cells by electroporation, a critical methodology for all researchers experimenting with embryonic stem cells. Replete with experimental detail, photo-micrographic documentation, and helpful technical pearls, this report walks the practitioner through the steps of cell culture, electroporation, drug selection, picking and expansion of colonies, cryopreservation and thawing of cell lines, DNA isolation for analysis, and preparation of cells for blastocyst injection or morula aggregation. The narrative is instructive without being preachy and will serve the curious experimentalist well.
We encourage and invite future efforts from those of you willing to share your experience and experimental wisdom, including narrative details that might not be found in protocol "handbooks." Manuscripts of considerable interest might include protocols for: A) preparation and characterization of stromal cell lines from various tissues and species that support embryonic and adult/somatic stem cell culture; B) manipulation of retroviral and lentiviral vectors and their expression patterns in mouse and human embryonic stem cells; C) differentiation and characterization of specific tissue lineages from mouse and human embryonic stem cells; D) primary isolation and culture of embryonic stem cells from mouse and other mammalian species; E) nuclear transfer and parthenogenesis, and F) obtaining approval from Institutional Review Boards for work with human embryos and embryonic stem cells. These are but a few of the possible topics that might pique the interest of the mounting wave of scientists eager to learn the latest techniques of embryonic stem cell manipulation. The community of biomedical scientists and the pace of scientific discovery will be well served by demystifying the methodology surrounding embryonic stem cells(George Q. Daley, M.D., Ph)