Fig. 2.

Characterization of the pseudo-attP sites in the mouse genome. (A) Integrated plasmids were rescued from total genomic DNA of transfected mouse 3T3 cells by bacterial cloning, and the flanking mouse DNA sequences were determined. The flanking mouse DNA sequences were compared with the available mouse genome sequence, and the corresponding phage integration sites were determined. These sites represent chromosomal locations of the pseudo-attP sties naturally occurring in the mouse genome. Eight different pseudo-attP sites were identified, which are located in mouse chromosomes 3, 5, 7, 4, 10, 9, and 8, respectively. All of these sites are located within the intergenic regions of the respective chromosomes. (B) Multiple alignments of the pseudo-attP sites in mouse genome. Compared with the wild-type attP sequence, the pseudo-attP showed only 35–50% sequence homology. The conservative regions are shown as shaded areas. (C) Integration frequency in vitro. To quantify the frequency of integration events at each pseudo-attP site, primers were designed to amplify the attL junctions. Standard dilution curves were established by using control plasmid DNAs of known concentrations. The data were normalized to genome copy number by using primers that amplify the single-copy mouse PAH gene per haploid genome. In 3T3 cells, >76% of integration events occurred at the major site mpsP3 (0.013 per haploid genome) and another 23% occurred in the minor sites mpsP5 and mpsP7 (0.0024 and 0.0017 per haploid genome). The integration frequencies on the other minor pseudo-attP sites were close to the detection limit.