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...

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...

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...

Highly Efficient BAC Modification Based on the R6Ky Origin of Replication

Bacterial Artificial Chromosome Vector

An important advance in the utilization of BACs for functional studies came with the development of methods for precise manipulation i.e., marker gene insertion, deletion, point mutation, etc. of BAC DNA Yang et al. 1997 . The ability to manipulate BAC constructs opened the door to strategies for gene expression and function studies, cell marking and isolation experiments that had previously been widely employed only in invertebrate systems Heintz 2000 . Several methods have been developed to...

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...