Controlled Genetic Manipulations

Abstract

The bacteriophage P1-derived Cre/ lox recombination system has been extensively used to engineer the genome of cultured cells and experimental animals. Cre recombinase recognizes the lox P site, which is composed of two 13-bp inverted repeats and an 8-bp spacer region, and mediates both intramolecular (excisive) and intermolecular (integrative) recombination between two lox P sites. The excision reaction is effi cient and can be used in conditional knockout strategies. On the other hand, integrative recombination is ineffi cient because the integrated DNA retains lox P sites at both ends and is easily excised again if the Cre recombinase is still present. However, integrative recombination is expected to be a powerful tool for genome engineering in mouse embryonic stem (ES) cells because it allows precise and repeated knock-in of any DNA into lox sites placed in the genome. To promote integrative recombination, two kinds of mutant lox systems have been developed and successfully used in ES cells to produce exchangeable (mul- tipurpose) alleles. In this chapter, we describe a Cre/mutant lox system for integrative recombination, and we present an application of this system to gene targeting. By incorporating mutant lox sites into gene targeting vectors, we can fi rst produce a null allele. Subsequently, any gene of interest, including the Cre recombinase gene itself, fl uorescent genes, luciferase genes, mutated cDNAs, and human cDNAs, can be inserted and expressed under the endogenous promoter of the targeted gene. By combining other recom- bination systems, such as Flp/ FRT , we can also convert null alleles into conditional alleles.