Gene Knockout Protocols

1. Front Matter

   Pages i-xvi

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2. Gene Modification in ES Cells

   1. Front Matter

      Pages 1-1

      PDF

   2. Overview on Mouse Mutagenesis

      • Ralf Kühn, Wolfgang Wurst

      Pages 1-12

   3. Construction of Gene-Targeting Vectors by Recombineering

      • Song-Choon Lee, Wei Wang, Pentao Liu

      Pages 15-27

   4. Gene-Trap Vectors and Mutagenesis

      • Silke De-Zolt, Joachim Altschmied, Patricia Ruiz, Harald von Melchner, Frank Schnütgen

      Pages 29-47

   5. Chromosome Engineering in ES Cells

      • Louise van der Weyden, Charles Shaw-Smith, Allan Bradley

      Pages 49-77

   6. 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 et al.

      Pages 79-99

   7. Generation of shRNA Transgenic Mice

      • Christiane Hitz, Patricia Steuber-Buchberger, Sabit Delic, Wolfgang Wurst, Ralf Kühn

      Pages 101-129

   8. Mutagenesis of Mouse Embryonic Stem Cells with Ethylmethanesulfonate

      • Robert Munroe, John Schimenti

      Pages 131-138

3. Stem Cell Manipulation

   1. Front Matter

      Pages 139-139

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   2. Gene Targeting in Mouse Embryonic Stem Cells

      • Lino Tessarollo, Mary Ellen Palko, Keiko Akagi, Vincenzo Coppola

      Pages 141-164

   3. Manipulating Mouse Embryonic Stem Cells

      • Eileen Southon, Lino Tessarollo

      Pages 165-185

   4. ES Cell Line Establishment

      • Heidrun Kern, Branko Zevnik

      Pages 187-204

   5. Generation of Double-Knockout Embryonic Stem Cells

      • Eva Wielders, Marleen Dekker, Hein te Riele

      Pages 205-218

   6. Differentiation Analysis of Pluripotent Mouse Embryonic Stem (ES) Cells In Vitro

      • Insa S. Schroeder, Cornelia Wiese, Thuy T. Truong, Alexandra Rolletschek, Anna M. Wobus

      Pages 219-250

   7. Cloning of ES Cells and Mice by Nuclear Transfer

      • Sayaka Wakayama, Satoshi Kishigami, Teruhiko Wakayama

      Pages 251-265

4. Genetically Engineered Mice

   1. Front Matter

      Pages 266-266

      PDF

   2. Isolation, Microinjection and Transfer of Mouse Blastocysts

      • Susan W. Reid, Lino Tessarollo

      Pages 269-285

   3. Aggregation Chimeras: Combining ES Cells, Diploid, and Tetraploid Embryos

      • Mika Tanaka, Anna-Katerina Hadjantonakis, Kristina Vintersten, Andras Nagy

      Pages 287-309

   4. VelociMouse: Fully ES Cell-Derived F0-Generation Mice Obtained from the Injection of ES Cells into Eight-Cell-Stage Embryos

      • Thomas M. DeChiara, William T. Poueymirou, Wojtek Auerbach, David Frendewey, George D. Yancopoulos, David M. Valenzuela

      Pages 311-324

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.

• DNA
• ES cells
• Gene modification
• Genetically engineered mice
• Mouse genome
• Mutagenesis techniques
• Mutant
• Phenotype analysis
• Stem cell manipulation
• chromosome
• cryopreservation
• genes
• genetics
• molecular biology

From the reviews of the second edition:

"This comprehensive collection of step-by-step protocols in the field of gene targeting and mouse genetics covers such topics as gene modification in embryonic stem cells … and mutant phenotype analysis. … The book will serve as a valuable resource for researchers, scientists, and students interested in these areas. … This is a well-illustrated book … . All researchers interested in gene targeting and gene knockout protocols will find this extremely useful." (Omer Iqbal, Doody’s Review Service, September, 2009)

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