Gene Knockout Protocols 2nd Edition by Wolfgang Wurst, Ralf Kühn 1934115266 9781934115268

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ISBN 10: 1934115266

ISBN 13: 9781934115268

Author: Wolfgang Wurst, Ralf Kühn

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 Knockout Protocols 2nd Table of contents:

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 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

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

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