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Describes techniques for the design of gene targeting constructs and for the analysis of the mouse phenotype, covering techniques of embryo transplantation, in vitro embryonic stem cell differentiation, creation of aggregation chimeras, mouse pathology, embryo cryopreservation, and transplantation. Protocols contain step-by-step instructions and notes on pitfalls to avoid. Of interest to biochemists, cell biologists, geneticists, and molecular biologists. Tymms is affiliated with the National Vision Research Institute, Australia. Kola is affiliated with Monash Medical Center, Monash University, Australia.Book News, Inc.?, Portland, OR


Book Info
National Vision Research Institute of Australia, Carlton, Victoria. Shows how gene targeting techniques can be used to simulate human disease states. Includes techniques for embryo transplantation and in vitro ES cell differentiation and offers instructions and notes on pitfalls to avoid. Outline format.

Preface v
Contributors ix
1 Overview: Gene Knockouts 1
2 Isolation and Maintenance of Primate Embryonic Stem Cells 11
3 Gene Targeting in ES Cells 19
4 Manipulating Mouse Embryonic Stem Cells 47
5 Gene Targeting in a Centralized Facility 65
6 The LoxP/CRE System and Genome Modification 83
7 Creation and Use of a Cre Recombinase Transgenic Database 95
8 Choice of Mouse Strains for Gene Targeting 107
9 Isolation, Microinjection, and Transfer of Mouse Blastocysts 121
10 Aggregation Chimeras: Combining ES Cells, Diploid and Tetraploid Embryos 135
11 How to Study Pathologic Phenotypes of Knockout Mice 155
12 Analysis of Hematopoietic Phenotypes in Knockout Mouse Models 181
13 Isolation of Embryonic Fibroblasts and Their Use in the In Vitro Characterization of Gene Function 205
14 Influence of Genetic Background on Knockout Mouse Phenotypes 217
15 Lineage Allocation During Early Embryogenesis: Mapping of the Neural Primordia and Application to the Analysis of Mouse Mutants 227
16 Generation of Double-Knockout Embryonic Stem Cells 251
17 In Vitro Differentiation of Embryonic Stem Cells and Analysis of Cellular Phenotypes 263
18 Embryonic Stem Cells in the Study of Hematopoiesis 287
19 Interferon-Inducible ES Cell Expression Systems 301
20 Transgenic Studies in the Mouse: Improving the Technology Towards a Conditional Temporal and Spatial Approach 319
21 In Vivo Libraries of Large Insert Transgenic Mice for Genetic Mapping 335
22 Epigenetic Effects on Transgene Expression 351
23 Positional-Candidate Cloning of Genes from Mouse Mutants 369
24 Genetically Engineered Mice: Husbandry and Resources 381
25 Embryo Cryopreservation for Transgenic Mouse Lines 397
Index