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. 2001 Jun;21(12):3926-34.
doi: 10.1128/MCB.21.12.3926-3934.2001.

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase

B Thyagarajan  1 , E C Olivares, R P Hollis, D S Ginsburg, M P Calos
Affiliations

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase

B Thyagarajan et al. Mol Cell Biol. 2001 Jun.

Abstract

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

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Figures

FIG. 1
FIG. 1
Plasmids used to detect chromosomal integration. (A) The pL-attB plasmid carried the φC31 attB site and the luciferase gene and was used to measure the level and persistence of luciferase expression in the presence and absence of the φC31 integrase, reflecting chromosomal integration at pseudo attP sites. A similar plasmid, pL-attP, carried the attP site and was used to monitor integration at pseudo attB sites. (B) pHZ-attP was used to place attP sites into the genome by using the hygromycin resistance selectable marker. pHZ-attB is a similar plasmid used for placement of attB sites in the genome. (C) pNC-attB, which carried an attB site and the neomycin resistance marker, was used as the incoming plasmid for identification of clones that underwent integration at an attP site or a pseudo attP site. pNC-attP was the parallel plasmid used to detect integration at attB or pseudo attB sites. (D) Zeocin selection scheme. From the top is shown a host chromosome (wavy lines) in which we integrated plasmid pHZ-attP (straight lines) bearing the hygromycin resistance gene for selection, an attP site for integrase-mediated recombination with attB, and a zeocin resistance gene lacking a promoter. The donor plasmid pNC-attB carries a neomycin resistance gene for selection, a green fluorescent protein (GFP) gene for analysis of transfection efficiency, and an attB site for integrase-mediated recombination with attP. Adjacent to attB is the strong promoter from CMV. Upon cotransfection of pNC-attB with pCMV-Int as a source of integrase, site-specific integration leads to the potential for expression of the zeocin resistance gene from the CMV promoter introduced on pNC-attB. The same donor plasmid was used in unmodified 293 and 3T3 cells, where it was selected with neomycin alone.
FIG. 2
FIG. 2
Luciferase expression after transfection of att-luciferase plasmids into unmodified 293 cells. Shown is the time course of luciferase expression for constructs that carried the luciferase gene and either no att site (pL), attB (pL-attB), or attP (pL-attP) and which were transfected with and without the integrase-expressing plasmid pCMV-Int. Each data point represents the average of three independent experiments, with appropriate standard error bars. A greatly elevated level of luciferase expression indicative of efficient chromosomal integration was seen only when the incoming plasmid carried an attB site and integrase was present. Similar data were obtained for mouse 3T3 cells.
FIG. 3
FIG. 3
DNA sequence characterization of pseudo attP sites and recombination junctions in human and mouse cells. (A) The wild-type φC31 attP site is shown on top. The 3-bp TTG crossover region is marked, and arrows delineate the imperfect palindromes. Beneath are the DNA sequences for four pseudo attP sites. The GenBank accession numbers for these pseudo attP sites are in parentheses below. Human ψA (AF333429) is the primary site of φC31 integrase-mediated integration in unmodified human cells, and human ψC (AF333430) and ψD (AF333431) are two secondary human integration sites. Mouse ψA (AF333432) is a site used repeatedly in mouse cells. A 100-bp DNA sequence centered on the attP core is presented here, with identities with attP shown filled in. (B) The sequences of the crossover region between the human ψA pseudo attP site (lowercase letters) and the attB site on the incoming plasmid (uppercase letters) for eight representative rescued events from neomycin-resistant pools of 293 cells. Colons represent bases missing in the novel joint. These small deletions ranged from 0 to 11 bp among 120 junctions sequenced. The bases affected by deletion in one or more junctions are highlighted in the composite junction sequence at the bottom of the diagram. (C) The sequences of the crossover region between an inserted wild-type attP site and the attB site on the incoming plasmid, which were derived from representative neomycin-resistant or neomycin- and zeocin-resistant colonies that were shown by PCR to have integrated at inserted attP site. The contribution from attP is shown in lowercase letters, while the contribution from attB and the 3-bp crossover region are in uppercase letters. The crossover junctions were perfect in each case (8 out of 8).
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