Candidate pig pseudo attP sites can reconstitute site-specific recombination in a functional rescue assay. (A) Scheme of reporter plasmid construction. Pig pseudo attP sites were cloned into pBCPB+ plasmid to replace the wild-type attP site by ABI-REC. The resultant plasmid was named p’BCPB+. This resulted in 4 p’BCPB+ plasmids, i.e. 5113, 5156, 1015, and 2015, respectively. p’BCPB+ was used to reconstitute the attB-pseudo attP recombination with the presence of PhiC31 integrase. For the ABI-REC details, please refer to additional files. (B) Rescue assay. PhiC31 integrase plasmid, pBCPB+, and 4 p’BCPB+ were transfected into PK15 cells. 48 h post transfection, genomic DNA was extracted and used for PCR detection of attR hybrid site with attR-F and attR-R primers. Pig endogenous MSTN gene (500 bp) was used as internal control to normalize the quantity of genomic DNA template. Primers attL-F and attR-R (555 bp) were used to quantify the amount of pBCPB+. (C) Recombination efficiency. The VisionCapture tool was used to quantify the density of the PCR product in (B). Recombination efficiency was calculated with the following formulation: Amount of attR/(amount of attR + amount of pBCPB+). It shows that the recombination efficiency of wild-type attP and attB sites was up to 80%. Pig 5113 pseudo attP site has a recombination efficiency of 60%, higher than the other 3 pig pseudo attP sites.