Figure 3

Confirmation of integration sites and allele analysis. (A) attR and attL detection by junction PCR. Integration site and its neighboring sequences were retrieved from UCSC Pig Genome. Primers were designed across the junction site. PCR analysis revealed that pEGFP-N1-attB had been integrated into the sites identified by TAIL-PCR. In addition, all the 4 integration events occurred to one chromosome as demonstrated, implying that the transgene is integrated as single-allele at the 4 pseudo attP sites. (B) Micro insertion or deletion at integration site. PCR products in Figure 3A were TA cloned and sequenced to analyze the integration fidelity. Micro insertion and deletion were observed in the integration sites, implying that DNA strand breakage and repair occurred during recombination. (C) Representative examples of micro insertion or deletion at integration site. For attR hybrid sequence of 5156 pseudo attP site, four nucleotides “ACCC” were inserted between PK15 cellular genome and transgene sequence. For attR of 2015 pseudo attP site, two nucleotides of attB were lost.