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Gene Knockout Protocols

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In this second edition, distinguished contributors with extensive experience in the gene targeting and mouse genetics fields revea... Weiterlesen
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In this second edition, distinguished contributors with extensive experience in the gene targeting and mouse genetics fields reveal a comprehensive set of step-by-step laboratory protocols and emphasize the new mutagenesis techniques developed in recent years.

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 phenotypegenotype 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.

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


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.

Chapter 1 Overview on Mouse Mutagenesis Ralf Kühn and Wolfgang Wurst Section I. Gene Modification in ES Cells Chapter 2 Construction of Gene Targeting Vectors by Recombineering Song-Choon Lee, Wei Wang, and Pentao Liu Chapter 3 Gene Trap Vectors and Mutagenesis Silke De-Zolt, Joachim Altschmied,, Patricia Ruiz, Harald von Melchner, and Frank Schnütgen Chapter 4 Chromosome Engineering in ES Cells Louise van der Weyden, Charles Shaw-Smith, and Allan Bradley Chapter 5 Gene Modification in Embryonic Stem Cells by Single-Stranded DNA Oligonucleotides Marieke Aarts, Marleen Dekker, Rob Dekker, Sandra de Vries, Anja van der Wal, Eva Wielders, and Hein te Riele Chapter 6 Generation of shRNA Transgenic Mice Christiane Hitz, Patricia Steuber-Buchberger,Sabit Delic, Wolfgang Wurst, and Ralf Kühn Chapter 7 Mutagenesis of Mouse Embryonic Stem Cells with Ethylmethanesulfonate Robert Munroe and John Schimenti Section II. Stem Cell Manipulation Chapter 8 Gene Targeting in Mouse Embryonic Stem Cells Lino Tessarollo, Mary Ellen Palko, Keiko Akagi, and Vincenzo Coppola Chapter 9 Manipulating Mouse Embryonic Stem Cells Eileen Southon and Lino Tessarollo Chapter 10 ES Cell Line Establishment Heidrun Kern and Branko Zevnik Chapter 11 Generation of Double Knockout Embryonic Stem Cells Eva Wielders, Marleen Dekker, and Hein te Riele Chapter 12 Differentiation Analysis of Pluripotent Mouse Embryonic Stem (ES) Cells in vitro Insa S. Schroeder, Cornelia Wiese, Thuy T.Truong, Alexandra Rolletschek, and Anna M. Wobus Chapter 13 Cloning of ES cells and Mice by Nuclear Transfer Sayaka Wakayama, Satoshi Kishigami, and Teruhiko Wakayama Section III. Genetically Engineered Mice Chapter 14 Isolation, Microinjection, and Transfer of Mouse Blastocysts Susan W. Reid and Lino Tessarollo Chapter 15 Aggregation Chimeras: Combining ES Cells, Diploid and Tetraploid Embryos Mika Tanaka, Anna-Katerina Hadjantonakis, Kristina Vintersten, and Andras Nagy Chapter 16 VelociMouse: Fully ES Cell-Derived F0 Generation Mice Obtained from the Injection of ES Cells into 8-Cell Stage Embryos Thomas M. DeChiara, William T. Poueymirou, Wojtek Auerbach, David Frendewey, George D. Yancopoulos, and David M. Valenzuela Chapter 17 Generation of Cre Recombinase Expressing Transgenic Mice Using Bacterial Artificial Chromosomes Jan Rodriguez Parkitna, David Engblom, and Günther Schütz Chapter 18 Inducible Cre Mice Susanne Feil, Nadejda Valtcheva, and Robert Feil Chapter 19 Creation and Use of a Cre Recombinase Transgenic Database Andras Nagy, Lynn Mar, and Graham Watts Chapter 20 Transposon Mutagenesis in Mice David A. Largaespada Chapter 21 Lentiviral Transgenesis Alexander Pfeifer and Andreas Hofmann Chapter 22 Sperm Cryopreservation and In Vitro Fertilization Susan Marschall, Auke Boersma, and Martin Hrabé de Angelis Section IV. Phenotype Analysis Chapter 23 Influence of Genetic Background on Genetically Engineered Mouse Phenotypes Thomas


Titel: Gene Knockout Protocols
EAN: 9781934115268
ISBN: 978-1-934115-26-8
Format: Fester Einband
Herausgeber: Springer, Berlin
Genre: Medizin
Anzahl Seiten: 516
Gewicht: 1288g
Größe: H33mm x B265mm x T203mm
Jahr: 2009
Auflage: 2nd ed.

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