Gene Knockout Protocols (Methods in Molecular Biology)

Following the completion of the mouse and human genome sequences, a major challengeisthefunctionalcharacterizationofeverymammaliangeneandthedeciph- ing of their molecular interaction network. The mouse offers many advantages for the use of genetics to study human biology and disease, unmatched among other m- mals. Its development, body plan, physiology, behavior, and diseases have much in common, based on the fact that 99% of the human genes have a mouse ortholog. The investigation of gene function using mouse models is based on many years of tech- logical development. In the two decades since gene targeting in murine embryonic stem (ES) cells was first described by Mario Capecchi and colleagues, more than 3000 predesigned mouse mutants have been developed. To date, a variety of mouse mutagenesis techniques, either gene- or phenotype-driven, are used as systematic approaches. The availability of the genome sequence supports gene-driven approaches such as gene-trap and targeted mutagenesis in ES cells, allowing efficient and precise gene disruption. In combination with the use of site-specific DNA recombinases, in particular the Cre/loxP system, gene disruptioncan be directed to specific cell types in conditionalmousemutants. Furthermore,chemicalandtransposonmutagenesisofthe mouse genome enables us to perform phenotype-driven screens for the unbiased identification of phenotype–genotype correlations involved in models of human d- ease. Over the next several years, the mouse genome will be systematically altered, and the techniques for achieving predesigned manipulations will be constantly developed further and improved. The second edition of Gene Knockout Protocols brings together distinguished c-tributorswithextensiveexperienceinthegenetargetingandmousegeneticsfields.

With the completion of the mouse and human genome sequences, the functional characterization of every mammalian gene and the deciphering of their molecular interaction network has become the next major challenge, and the mouse genome provides a convenient and vital model for these studies. In Gene Knockout Protocols, Second Edition, distinguished contributors with extensive experience in the gene targeting and mouse genetics fields reveal a comprehensive collection of step-by-step laboratory protocols. Emphasizing the many new mutagenesis techniques developed over the last seven years, the volume covers topics such as gene modification in ES cells, stem cell manipulation, the generation of genetically engineered mice, and mutant phenotype analysis. Written in the highly successful Methods in Molecular Biology™ series format, chapters contain brief introductions to their respective subjects, lists of the necessary materials and reagents, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Fully updated and authoritative, Gene Knockout Protocols, Second Edition provides a unique collection of expert bench protocols certain to be a valuable resource for those just entering the field of gene targeting but also for all genetic researchers today.