Practical Approach Series: Gene Targeting

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The book details the rationale behind designing gene targeting vectors, focusing on basic replacement vectors for null mutations and advanced two-step methods for subtle mutations. It covers the latest techniques for culturing and manipulating embryonic stem (ES) cells, as well as approaches for identifying rare targeted cell clones. The text discusses culturing and assaying mouse and human hematopoietic progenitors both in vitro and in vivo, and outlines the design and use of retrovirus vectors for gene introduction and expression in bone marrow cells. Two methods for creating ES cell chimeras are presented: the standard approach of injecting ES cells into blastocysts and morula injection, alongside techniques for making aggregation chimeras, particularly using tetraploid host embryos to generate entirely ES cell-derived embryos and mice. Critical steps for achieving germline chimeras are also examined. The book concludes with a discussion on the various applications of enhancer, gene, and promoter trap vectors, detailing the advantages of each and the techniques for producing and analyzing "trap" vector insertions in ES cells. The protocols are designed to be accessible for novices, serving as a manual for producing mutant mice, from embryonic stem cells to chimeric mice suitable for breeding.