29] Targeted Inactivation of the G12α gene with replacement and insertion vectors: Analysis in a 96-well plate format

Abstract

This chapter investigates the targeted inactivation of the G i2 α gene with replacement and insertion vectors. It has become possible to inactivate genes in mammalian cells by introducing a mutation at a specific genomic locus. Murine embryonic stem cells (ES cells) have been isolated that can be genetically manipulated in cell culture and reintroduced into blastocysts, giving rise to chimeric animals. Chimeras may transmit the mutation to their offspring, which will then be heterozygous for the mutation. The mutation can then be bred to homozygosity, enabling the analysis of the mutational phenotype both at the level of the whole animal and at the cellular and molecular level by analyzing cell lines or tissues derived from animals homozygous for the desired mutation. An important task in gene targeting experiments is to identify the rare targeted events over the background of random integrations. Most protocols include the use of a Neo cassette in the targeting vector disrupting an exon of the gene of interest and enabling a positive selection for cells that have stably integrated the transfected DNA into the genome.