Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Science, Wuhan 430064, China.
BMC Mol Biol. 2013 Sep 8;14:20. doi: 10.1186/1471-2199-14-20.
Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5' and 3' junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.
PhiC31 整合酶以单向特异性方式将含有 attB 的质粒整合到真核基因组中的假 attP 位点，并维持强大的转基因表达。然而，很少有研究探索其在畜牧业中的潜力。本研究旨在发现 PhiC31 整合酶介导的猪细胞特异性重组的分子基础。我们表明，PhiC31 整合酶可以介导转基因特异性整合到猪肾 PK15 细胞的基因组中。瞬时转染含有 attB 和 attP 的质粒时，猪 PK15 细胞系中的分子内重组发生的最大频率为 82%。在药物选择下，观察到 pCMV-Int 与 pEGFP-N1-attB 的最佳摩尔比为 5:1 时，细胞克隆数量最多。从那些单拷贝转基因整合的细胞克隆中，通过 TAIL-PCR 鉴定了四个候选假 attP 位点。其中两个发生了更高的整合频率，为 33%。5'和 3' 连接 PCR 表明，PhiC31 整合酶介导的转基因整合是单等位基因的。通过整合边界测序观察到微缺失和插入，表明重组诱导了双链断裂。然后，我们通过将四个假 attP 位点的 ABI-REC 克隆到 pBCPB+ 质粒中，构建了挽救报告质粒。这些挽救质粒和 pCMV-Int 的转染导致 attB 和假 attP 位点之间发生预期的分子内重组。这证明了内源性假 attP 位点是 PhiC31 整合酶介导的特异性重组的功能性底物。两个假 attP 位点维持了强大的细胞外和细胞内 EGFP 表达。Alamar blue 测定表明，将转基因整合到这些特定位点对细胞增殖几乎没有影响。这是第一个报道 PhiC31 整合酶在猪细胞中介导特异性重组的潜在用途的报告。我们的工作建立了一个理想的模型来研究位于不同染色体环境中的相同转基因的位置效应。这些发现也为靶向猪基因组工程奠定了基础，并可用于生产用于农业和生物医学用途的转基因猪。
