Chromosomal Mutagenesis

Volume 435 of the series Methods in Molecular Biology pp 165-173

Site-Specific Chromosomal Integration Mediated by ϕC31 Integrase

  • Annahita KeravalaAffiliated withDepartment of Genetics, Stanford University School of Medicine
  • , Michele P. CalosAffiliated withDepartment of Genetics, Stanford University School of Medicine

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ϕC31 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 ϕC31 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 ϕC31 integrase supplies a simple method to obtain repeated integration at the same chromosomal site in mammalian cells.

Key Words

attB attP ϕC31 integrase phage integrase position effects site-specific integration