Contents

1 Generation of Transgenic Animals Using Lentiviral Vectors 1 1.2 The Production of Lentiviral of the Lentiviral Transfer 1.2.4 Plasmid 1.2.5 Preparation of 293T 1.2.7 Viral 1.2.8 Virus 1.3 Production of Transgenic Mice and 1.3.1 Superovulation and Embryo 1.3.2 Delivery of Lentiviruses to Single-Cell Embryos 14 1.3.3 Transfer of Embryos into Recipient 1.4 Establishment of Stable Strains from Lentiviral Founder 1.4.1 Mating of Founder 1.5 Safety Guidelines for Pseudotyped References 2...

Derivation of Nuclear Transfer ES Cells

The methods we use for derivation of ES cell lines from NT embryos are similar to those previously reported (Hogan et al. 1994) with several modifications (Hochedlinger and Jaenisch 2002a Eggan et al. 2004). Following oocyte activation, NT embryos are cultured in vitro in KSOM for 3-4 days. Embryos reaching the morula or blastocyst stage after this time are used for ES cell derivation. NT morulae and blastocysts are exposed to acid Ty-rode's solution (Specialty Media) to remove the zona...

Quality and Treatment of Sperm Used for ICSI

Probably the single most important justification for the development and application of ICSI for mouse colony management is the minimal requirement of sperm amenable to ICSI. Sperm from a male mouse that is fertile, subfertile, or infertile, including a male that either physically cannot mate or cannot produce normal sperm capable of fertilizing oocytes naturally either in vivo or in vitro, can be used successfully for ICSI. Sperm types that have been used successfully include epididymal and...

Preface

Now that the genomes of several animal species have been sequenced, the need to manipulate the genome in order to understand gene function has stimulated an even wider field of research. The impact of the techniques of gene manipulation on the understanding of developmental biology and disease in humans, as well as in other species, is exciting. Since the first transgenic animal was made by Palmiter and Brinster in 1982 and the first targeted mutation by Capecchi, Evans and Smithies in 1989,...

ICSI Instrumentation

Despite the very successful and multiple applications of ICSI as an effective ART in mice, the complex nature of the procedure and specialized instrumentation required have been cited by some as making ICSI inaccessible to most workers in the field, with the exception of a few highly specialized laboratories. These are unfair assertions, as the instrumentation differs in only a few respects from that used in most transgenic laboratories today, and the procedure can be mastered with concerted...

Advantages of Using Tetraploid Embryo Complementation

Tetraploid Complementation

The production of genetically engineered mice by conventional methods is highly time consuming, and involves the generation of mutant alleles in ES cells by homologous recombination, the production and breeding of chimeric founder mice and, finally, crosses between the resulting heterozygous mice to produce homozygous mutant offspring Fig. 3.2A . The primary advantage of using tetraploid-embryo complementation in the pro duction of mutant mice is that the heterozygous mice can be produced...