A phage integrase directs efficient site-specific integration in human cells - PubMed (original) (raw)
A phage integrase directs efficient site-specific integration in human cells
A C Groth et al. Proc Natl Acad Sci U S A. 2000.
Abstract
The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.
Figures
Figure 1
Schematic diagrams of five plasmids used in demonstrating function of the φC31 integrase. (A) pInt, a plasmid for expression of φC31 integrase in E. coli. (B) pCMVInt, for expression of integrase in mammalian cells. (C) pBCPB+, intramolecular integration assay vector. (D) p220KattBfull, an EBV vector bearing attB, the target for integration events. (E) pTSAD, the donor for integration events, bears attP. KanR, AmpR, ChlorR, HygR, genes for resistance to kanamycin, ampicillin, chloramphenicol, and hygromycin, respectively; SV40, simian virus 40; EBNA-1, Epstein–Barr virus-encoded nuclear antigen 1.
Figure 2
PCR analysis of recombination in human cells. A 401-bp product is indicative of site-specific recombination. Lane 1, untransfected pBCPB+; lane 2, _in vitro_-reacted pBCPB+ (see Materials and Methods); lane 3, Hirt extract (12) containing transfected pBCPB+ from human 293 cells; lane 4, Hirt extract containing transfected pBCPB+ and pCMVInt from human 293 cells; lanes M, size markers.
Figure 3
Minimal sizes of attB and attP sites. The right column shows the percentage recombination obtained in the intramolecular integration assay in E. coli when the illustrated shortened versions of φC31 attB (Upper) and attP (Lower) were tested. The name of each site tested corresponds to the length of the att site in base pairs. B33A and B33B indicate sites where the reduction of the site length from 34 bp to 33 bp occurred at the right or left ends of the site, respectively. Similar nomenclature is used for P39A and P39B. full attP, full-length attP.
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