In this paper we demonstrated the catalyzing activity of PhiC31 integrase to introduce site-specific transgene integration into PK15 cellular genome. We showed that PhiC31 integrase system work synergistically in a quantitative manner. For the extra chromosomal site-specific recombination, it was found that the optimal ratio of pCMV-Int (6230bp) to pBCPB+ (7221bp) is 5:1 in pig cellular environment. PhiC31 integrase catalyzes the recombination as high as 82%. In an attempt to examine the potency of PhiC31 integrase-mediated intermolecular recombination, we also observed that a 5:1 molar ratio of pCMV-Int to pEGFP-N1-attB resulted in robust and long term EGFP expression and produced maximum percentage of EGFP-positive cell clones. Please note that in all the transfections we have assured the charge ratio of DNA plasmid to transfection reagent invariable. Hence, our results allow for a direct comparison among diverse transfection settings. Such results were also observed in previous reports [17, 20]. Exact explanations of the “overproduction-inhibition effect” remain unknown. But careful examination of molar ratio of enzyme to substrate is a critical step prior to its successful use. This also suggests that, given that an unsuitable enzyme/substrate ratio is used, the efficacy of this system will be depressed.
Secondly, as documented previously in human, mouse, rat, fruit fly, silkworm and cattle, pseudo attP site profile varies across species . Four pseudo attP sites were mapped in PK15 cellular genome in this work. All of them were located in intergenic regions, and none was found in other mammalian genomes. This indicates that PhiC31 integrase-mediated transgene integration pattern is species-specific, and the sites identified in this work are unique to pig. Our findings also suggest that pseudo attP sites in mammalian genome are not conserved during evolution. Additionally, this is unlike the integration pattern from virus gene transfer system, such as MLV that shows a strong preference for transcription start sequences . In this regard, PhiC31 integrase functions in a safe way to modify host genome. On one hand, PhiC31 integrase is not likely to induce endogenous gene mutagenesis in pig cells. On the other hand, we did not observe any aberrant morphology and abnormal proliferation in the transgenic cell lines. Thus they are in favor of transgene integration and expression. Especially, 5113 and 5156 pseudo attP sites are ideal locus for robust transgene expression. This is particularly advantageous to gene therapy and safe transgenic technology, as this will reduce the risk of insertional mutagenesis [25, 26].
Thirdly, we reconstituted the site-specific recombination by performing a functional rescue assay. To our knowledge, this is the first report to prove candidate pseudo attP site to be bona fide attP site. Through this test, we ruled out the possibility that pEGFP-N1-attB happened to be randomly integrated into candidate pig pseudo attP sites. This also indicates that the recognition site of PhiC31 integrase in PK15 cellular genome is not stringent, as the maximum similarity to wild type attP is around 30%. We observed an inverse correlation between similarity and recombination efficiency. It implies that there might be certain endogenous factors affect the sequence specificity of PhiC31 integrase . But this needs a further research of binding proteins interacting with PhiC31 integrase. Hence, improvement of its recognition specificity is necessary in future studies to prevent aberrant recombination and multiple site integration .
Finally, we carried out TAIL-PCR to isolate the integration site. In previous studies, inverse PCR, half-nested PCR and plasmid rescue were often used to identify the candidate pseudo attP sites. But the common drawback of these methods is that they require a large starting amount of genomic DNA (usually 10 μg) for restriction and ligation. However, TAIL-PCR needs as little as 0.5 μg genomic DNA. In this work, we obtained four pig pseudo attP sites by TAIL-PCR. Moreover, the integration of reporter plasmid into all these four pseudo attP sites could be proved by junction PCR, indicating that the positive rate of TAIL-PCR is 100%. Accordingly, we believe that TAIL-PCR is a valuable tool owing to its ease, low cost and high reproducibility.