Gene Knockout Protocols (2^nd Ed., 2nd ed. 2009) Methods in Molecular Biology Series, Vol. 530

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.

Gene Modification in ES Cells.- Overview on Mouse Mutagenesis.- Construction of Gene-Targeting Vectors by Recombineering.- Gene-Trap Vectors and Mutagenesis.- Chromosome Engineering in ES Cells.- Gene Modification in Embryonic Stem Cells by Single-Stranded DNA Oligonucleotides.- Generation of shRNA Transgenic Mice.- Mutagenesis of Mouse Embryonic Stem Cells with Ethylmethanesulfonate.- Stem Cell Manipulation.- Gene Targeting in Mouse Embryonic Stem Cells.- Manipulating Mouse Embryonic Stem Cells.- ES Cell Line Establishment.- Generation of Double-Knockout Embryonic Stem Cells.- Differentiation Analysis of Pluripotent Mouse Embryonic Stem (ES) Cells In Vitro.- Cloning of ES Cells and Mice by Nuclear Transfer.- Genetically Engineered Mice.- Isolation, Microinjection and Transfer of Mouse Blastocysts.- Aggregation Chimeras: Combining ES Cells, Diploid, and Tetraploid Embryos.- VelociMouse: Fully ES Cell-Derived F0-Generation Mice Obtained from the Injection of ES Cells into Eight-Cell-Stage Embryos.- Generation of Cre Recombinase-Expressing Transgenic Mice Using Bacterial Artificial Chromosomes.- Inducible Cre Mice.- Creation and Use of a Cre Recombinase Transgenic Database.- Transposon Mutagenesis in Mice.- Lentiviral Transgenesis.- Sperm Cryopreservation and In Vitro Fertilization.- Phenotype Analysis.- Influence of Genetic Background on Genetically Engineered Mouse Phenotypes.- Pathologic Phenotyping of Mutant Mice.- Systemic First-Line Phenotyping.

Comprehensive guide to all techniques required for the generation of classical and conditional knockout mouse strains Includes techniques for ES cell establishment, ES cell in vitro differentatio, and nuclear cloning Provides a systematic overview of techniques for mutant phenotyping Covers all gene knockout techniques, including gene targeting, chemical, gene trap, and transposon mutagenesis Written by hands-on experts who have optimized each method for experimental success