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This laboratory manual, published in cooperation with the International Society for Transgenic Technology (ISTT), provides almost all current methods that can be applied to the creation and analysis of genetically modified animals. The chapters have been contributed by leading scientists who are actively using the technology in their laboratories. Based on their first-hand experience the authors also provide helpful notes and troubleshooting sections. It includes topics that range from standard techniques, such as pronuclear microinjection of DNA, to more sophisticated and modern methods, such as the derivation and establishment of embryonic stem (ES) cell lines, with defined inhibitors in cell culture medium. In addition, related topics with relevance to the field are addressed, including global web-based resources, legal issues, colony management, shipment of mice and embryos, and the three R's: refinement, reduction and replacement.
Contents
1 Patent and Licensing Issues in Transgenic Technology
2 Global Resources: Including Gene Trapped ES Cell Clones - Is Your Gene Already Knocked Out?
3 Designing Transgenes for Optimal Expression
4 Gene Targeting Vector Design for Embryonic Stem Cell Modifications
5 Transgenic Production Benchmarks
6 Generation of Transgenic Mice by Pronuclear Microinjection
7 Generation of Transgenic Rats Using Microinjection of Plasmid DNA or Lentiviral Vectors
8 Generation of Transgenic Animals by Use of YACs
9 BAC Transgenes, DNA Purification, and Transgenic Mouse Production
10 Generation of Transgenic Animals with Lentiviral Vectors
11 Vertebrate Transgenesis by Transposition
12 Rat Spermatogonial Stem Cell-Mediated Gene Transfer
13 Mouse Cloning by Nuclear Transfer
14 Gene Targeting in Embryonic Stem Cells
15 The Importance of Mouse ES Cell Line Selection
16 Tetraploid Complementation Assay
17 Combining ES Cells with Embryos
18 Derivation of Murine ES Cell Lines
19 Rat Embryonic Stem Cell Derivation and Propagation
20 Induced Pluripotency: Generation of iPS Cells from Mouse Embryonic Fibroblasts
21 The Preparation and Analysis of DNA for Use in Transgenic Technology
22 Colony Management
23 Cryopreservation
24 Shipment of Mice and Embryos
25 Pathogen-Free Mouse Rederivation by IVF, Natural Mating and Hysterectomy
26 Refinement, Reduction, and Replacement
Index
