Isolation of pig pseudo attP sites. (A) Screening of transgenic cell lines with single-copy transgene integration by quantitative PCR. 34 transgenic cell lines expressing EGFP (shown in x-axis) were screened for single-copy transgene integration within PK15 cellular genome. This assay demonstrated that 11 cell lines harbored a single-copy pEGFP-N1-attB. The other 23 cell lines had distinct copy numbers of pEGFP-C1-attB, varying from 2 copies to 15 copies. (B) Isolation of integration sites. Left, microscopic images of four transgenic cell clones under fluorescence field (10×). Middle, TAIL-PCR was performed to isolate the integration sites. Right, Specific PCR products were TA cloned, sequenced and aligned by BALT against pig genome (UCSC). Red arrow represents pig genome sequence, and green arrow represents pEGFP-N1-attB sequence. The lanes shown as 1st, 2nd and 3rd represent the first, second and third round TAIL-PCR products fractionized by 1.5% agarose gel. M is 1 kb DNA molecular marker (Fermentas).