Overview -- Isolation and Maintenance of Primate Embryonic Stem Cells -- Gene Targeting in ES Cells -- Manipulating Mouse Embryonic Stem Cells -- Gene Targeting in a Centralized Facility -- The LoxP/CRE System and Genome Modification -- Creation and Use of a Cre Recombinase Transgenic Database -- Choice of Mouse Strains for Gene Targeting -- Isolation, Microinjection, and Transfer of Mouse Blastocysts -- Aggregation Chimeras -- How to Study Pathologic Phenotypes of Knockout Mice -- Analysis of Hematopoietic Phenotypes in Knockout Mouse Models -- Isolation of Embryonic Fibroblasts and Their Use in the In Vitro Characterization of Gene Function -- Influence of Genetic Background on Knockout Mouse Phenotypes -- Lineage Allocation During Early Embryogenesis -- Generation of Double-Knockout Embryonic Stem Cells -- In Vitro Differentiation of Embryonic Stem Cells and Analysis of Cellular Phenotypes -- Embryonic Stem Cells in the Study of Hematopoiesis -- Interferon-Inducible ES Cell Expression Systems -- Transgenic Studies in the Mouse -- In Vivo Libraries of Large Insert Transgenic Mice for Genetic Mapping -- Epigenetic Effects on Transgene Expression -- Positional-Candidate Cloning of Genes from Mouse Mutants -- Genetically Engineered Mice -- Embryo Cryopreservation for Transgenic Mouse Lines.

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.