Site-specific chromosomal integration mediated by C31 integrase

Abstract

C31 integrase is a site-specific recombinase from a bacteriophage that has become a useful tool in mammalian cells. The enzyme normally performs precise, unidirectional recombination between two attachment or att sites called attB and attP. We have shown that an attP site preintegrated into a mammalian chromosome can serve as a target for integration of an introduced plasmid carrying an attB site. Recombination leads to precise integration of the plasmid into the chromosome at the attP site. This reaction is useful for placing introduced genes into the same chromosomal environment, in order to minimize position effects associated with random integration. Because C31 integrase can also mediate integration at endogenous sequences that resemble attP, called pseudo attP sites, a selection system is used that yields integration only at the desired preintegrated attP site. This chapter provides a protocol that features a simple antibiotic selection to isolate cell lines in which the introduced plasmid has integrated at the desired attP site. A polymerase chain reaction assay is also presented to verify correct chromosomal placement of the introduced plasmid. This integration system based on C31 integrase supplies a simple method to obtain repeated integration at the same chromosomal site in mammalian cells. © 2008 Humana Press Inc.