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Over the past ten years it has become possible to make essentially any mutation in the germline of mice by utilizing homologous recombination and embryonic stem (ES) cells. As the era of gene discovery comes to an end, in vivo approaches for studying gene function will be utilized to an even greater extent. This new edition of Gene Targeting. A Practical Approach contains a remarkable collection of state-of-the-art protocols as well as thoughtful discussion of how to design a new mouse mutation and analyse the resulting phenotype to maximise the information gained. The techniques described for gene targeting in mouse ES cells, are equally applicable for gene targeting in other tissue culture cells, including human cells. The second edition describes additional genetic approaches for studying gene function in mice, including chimera analysis, gene trap (GT) approaches and powerful ways to combine gene targeting with classical genetics.
The book starts by describing the design of gene targeting vectors for mammalian cells and how to distinguish random integrations from homologous recombination. Topics then covered include: extending conventional gene targeting manipulations by site-specific recombination using the Cre-loxP and Flp-FRT systems to produce germline mutations that involve changing only a few base pairs or conditional (in)activation of genes; methods for introducing mutations into ES cells via homologous recombination including selection and screening procedures for identifying and recovering targeted cell clones; the establishment of new ES cell lines; aggregation versus blastocyst injection to create mouse chimeras and study mutant phenotypes; GT strategies including the components, design, and modification of GT vectors and screens; the molecular analysis of GT integrations; the use of classical genetics in studying targeted mutations and phenotype analysis.
Gene Targeting: A Practical Approach (second edition) is an indispensable practical tool and information resource for scientists working on fundamental or applied aspects of gene targeting, ES cell manipulation, and analysis of mouse mutants. It will be invaluable to all researchers who wish to use the approaches for studies of gene function in cells or mice.