UNIT 4.29 Overview of Gene Targeting by Homologous Recombination

The analysis of mutant organisms and cell lines is important in determining the function of specific proteins. Recent technological advances in gene targeting by homologous recombination in mammalian systems enable the production of mutants in any desired gene, and can be used to produce mutant mouse strains and mutant cell lines. The yeast Flp/FRT recombinase system and bacteriophage recombinases such as Cre and its recognition sequence, loxP, allow spatial and temporal control of knockouts. This unit discusses crucial issues for homologous recombination experiments, including requirements for the source of DNA, criteria for the targeting constructs, methods of enrichment for homologous recombinants, (positive and negative selection, and the use of endogenous promoters), and the types of mutations that can be created. Curr. Protoc. Neurosci. 40:4.29.1-4.29.13. © by John Wiley & Sons, Inc.

Keywords: Cre-loxP; FlP/FRT; recombinase; mammalian; phenotypic selection; homologous recombination; mutation