Gene Targeting

Gene targeting is a relatively new area of research, and no comprehensive manual exists which specifically addresses producing mutant mice from embryonic stem cells. This book focuses on using gene targeting to produce mutant mice since this is the most popular and perhaps most powerful approach, however gene trap approaches and the use of tetraploid aggregation chimeras for analyzing mutant ES cells in vivo are also included. It should also be stressed that the protocols are so detailed that they can be used as a manual. The relevant parts of the two competing books don't come anywhere close to the degree of detail. An aim of this book was that novices could use the book alone to go from ES cells to mutant mice. The book begins by describing in detail the rationale behind designing gene targeting vectors, including both basic replacement vectors for creating null mutations and more sophisticated two-step approaches for creating subtle mutations. It then goes on to describe the latest techniques for culturing and manipulating ES cells and a number of approaches for identifying rare targeted cell clones.
Techniques for culturing and assaying for mouse and human haematopoietic progenitors in vitro and in vivo are then covered, followed by the design of, and use of, retrovirus vectors for introducing and expressing genes in bone marrow cells. Two approaches for making ES cell chimeras are described. One chapter covers the more standard approach of ES cell injection into blastocysts, as well as morula injection. Another chapter describes techniques for making aggregation chimeras and in particular a recent approach using tetraploid host embryos to produce completely ES cell-derived embryos and mice. The critical steps for obtaining germline chimeras are disussed. The book is concluded by a discussion of the many uses of enhancer, gene and promoter trap vectors, the advantages of each, and describes in detail the techniques for producing and analyzing "trap" vector insertions in ES cells. The protocols are written with sufficient detail for a novice in the area to use them as a manual to learn the various techniques required for producing mutant mice, beginning with the embryonic stem cells and ending up with chimeric mice for breeding.