PhiC31 integrase is active in pig cells. (A) Mechanism of action of PhiC31 integrase-mediated site-specific recombination. The integrase catalyzes the intramolecular recombination between attB and attP sites within a single plasmid, producing two mini-circle plasmids with hybrid attR and attL sites, respectively. (B) PhiC31 integrase is functional in pig cells. Transfection of pig PK15 cells were performed with three different ratios of PhiC31 integrase plasmid to reporter pBCPB+. Resultant attL and attR sites were examined by PCR. The indicated recombination efficiency was calculated by measuring signal intensity in Visioncapture with the following formula: recombination efficiency = Intensity of attL/Intensity of (attL and pBCPB + backbone). A 5:1 ratio had 82% recombination efficiency. (C) The map of pEGFP-N1-attB (5138 bp). A 400 bp attB sequence was sub-cloned into the AseI site of pEGFP-N1, resulting in the site-specific integration reporter pEGFP-N1-attB. (D) EGFP expression level in a time lapse. The y-axis shows the expression levels and x-axis shows days after transfection. Four groups of plasmids (pEGFP-N1, pEGFP-N1-attB, pEGFP-N1/PhiC31 and pEGFP-N1-attB/PhiC31) were transfected into PK15 cells. EGFP expression level was quantified by measuring the extracellular EGFP by a Glomax Multi Jr fluorescence detector. IGF-I was detected by ELISA and used as an internal control for data normalization. pEGFP-N1-attB/PhiC31 maintained high EGFP level in comparison to other settings. (E) An optimal molar ratio of PhiC31 and pEGFP-C1-attB plasmids produced more cell clones expressing EGFP. Various ratios of PhiC31 and pEGFP-N1-attB plasmids were transfected into PK15 cells and cell clones were selected by G418. The y-axis shows % of green to total clones and x-axis shows different molar ratios. A 5:1 ratio produced more cell clones expressing EGFP, significant higher than other settings, including random integration (0/1 ratio). *, p < 0.01.
Bi et al. BMC Molecular Biology 2013 14:20 doi:10.1186/1471-2199-14-20