Gene Knockout Protocols

ISBN-13: 9781617370809 / Angielski / Miękka / 2010 / 431 str.

Martin J. Tymms; Ismail Kola

Opis

As the major task of sequencing the human genome is near completion and full complement of human genes are catalogued, attention will be focused on the ultimate goal: to understand the normal biological functions of these genes, and how alterations lead to disease states. In this task there is a severe limitation in working with human material, but the mouse has been adopted as the favored animal model because of the available genetic resources and the highly conserved gene conservation linkage organization. In just of ten years since the first gene-targeting experiments were p- formed in embryonic stem (ES) cells and mutations transmitted through the mouse germline, more than a thousand mouse strains have been created. These achievements have been made possible by pioneering work that showed that ES cells derived from preimplantation mouse embryos could be cultured for prolonged periods without differentiation in culture, and that homologous rec- bination between targeting constructs and endogenous DNA occurred at a f- quency sufficient for recombinants to be isolated. In the next few years the mouse genome will be systematically altered, and the techniques for achi- ing manipulations are constantly being streamlined and improved.

Recenzje

"This is a valuable reference work, which will no doubt find a home on many lab benches." -E-STREAMS

Spis treści

Overview: Gene Knockouts, Paul J. Hertzog and Ismail Kola. Isolation and Maintenance of Primate Embryonic Stem Cells, Vivienne S. Marshall, Michelle A. Waknitz, and James A. Thomson. Gene Targeting in ES Cells, Thomas M. DeChiara. Manipulating Mouse Embryonic Stem Cells, Lino Tessarollo. Gene Targeting in a Centralized Facility, Louise D. Barnett and Frank Köntgen. The LoxP/CRE System and Genome Modification, Trevor J. Wilson and Ismail Kola. Creation and Use of a Cre Recombinase Transgenic Database, Andras Nagy and Lynn Mar. Choice of Mouse Strains for Gene Targeting, Mireille H. Lahoud, Trevor J. Wilson, and Paul J. Hertzog. Isolation, Microinjection, and Transfer of Mouse Blastocysts, Angelika Bonin, Susan W. Reid, and Lino Tessarollo. Aggregation Chimeras: Combining ES Cells, Diploid and Tetraploid Embryos, Mika Tanaka, Anna-Katerina Hadjantonakis, and Andras Nagy. How to Study Pathologic Phenotypes of Knockout Mice, Roderick T. Bronson. Analysis of Hematopoietic Phenotypes in Knockout Mouse Models, Ivan Bertoncello and Brenda Williams. Isolation of Embryonic Fibroblasts and Their Use in the In Vitro Characterization of Gene Function, Paul J. Hertzog. Influence of Genetic Background on Knockout Mouse Phenotypes, L. Philip Sanford, Suhas Kallapur, Ilona Ormsby, and Thomas Doetschman. Lineage Allocation During Early Embryogenesis: Mapping of the Neural Primordia and Application to the Analysis of Mouse Mutants, Gabriel A. Quinlan, Bruce P. Davidson, and Patrick P. L. Tam. Generation of Double-Knockout Embryonic Stem Cells, Hein te Riele, Conny Brouwers, and Marleen Dekker. In Vitro Differentiation of Embryonic Stem Cells and Analysis of Cellular Phenotypes, Anna M. Wobus, Kaomei Guan, and Uta Pich. Embryonic Stem Cells in the Study of Hematopoiesis, Francesca Lazner, Ismail Kola, Elizabeth Stadler, and Andrew G. Elefanty. Interferon-Inducible ES Cell Expression Systems, Linda M. Whyatt and Peter D. Rathjen. Transgenic Studies in the Mouse: Improving theTechnology Towards a Conditional Temporal and Spatial Approach, Sika Ristevski. In Vivo Libraries of Large Insert Transgenic Mice for Genetic Mapping, Desmond J. Smith. Epigenetic Effects on Transgene Expression, Emma Whitelaw, Heidi Sutherland, Margot Kearns, Hugh Morgan, Linda Weaving, and David Garrick. Positional-Candidate Cloning of Genes from Mouse Mutants, Karen B. Avraham. Genetically Engineered Mice: Husbandry and Resources, John J. Sharp, Carol C. Linder, and Larry E. Mobraaten. Embryo Cryopreservation for Transgenic Mouse Lines, Jillian M. Shaw and Magosaburo Kasai. Index.

Blurb

As the major task of sequencing the human genome nears completion and the full complement of human genes are catalogued, the task of understanding the normal biological functions of genes and how their alteration leads to diseased states becomes more imperative. In Gene Knockout Protocols, highly skilled investigators with extensive experience in gene targeting and mouse genetics describe their best techniques for the design of targeting constructs and for genetic phenotype analysis. These proven methods contain step-by-step instructions, as well as notes on pitfalls to avoid, and emphasize techniques that are relevant to researchers carrying out gene targeting work. These include embryo transplantation, in vitro embryonic stem cell differentiation, creation of aggregation chimeras, mouse pathology, embryo cryopreservation, and transplantation. Issues such as the use of existing mouse mutation resources and the influence of genetic background and epigenetic effects upon phenotype are also covered.
State-of-the-art and highly practical, Gene Knockout Protocols not only constitutes an invaluable source of readily reproducible techniques for those just entering the field of gene targeting, but also a key reference for all genetic researchers today.