ΦC31整合酶介导猪基因组定点修饰的探讨

高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的"友好位点".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.

Site-specific Modification of Pig Genome Mediated by ΦC31 Integrase

Efficient and targeted genome modification is now the hot topic in gene engineering animal. Streptomyces ΦC31 integrase is capable of integrating attB-containing plasmid into pseudo attP site in mammalian genomes, resulting in the normal structure and high expression of transgene. This study is to discover the molecular basis of ΦC31 integrase-mediated site-specific transgene integration in pig genome. A reporter plasmid pEGFP-N1-attB was constructed and co-transfected with ΦC31 integrase-expressing plasmid pCMV-INT into pig kidney PK15 cells. Cell clones were generated under G418 selection. Quantitative real time PCR was used to identify cell clones with single-copy transgene integration. One pig pseudo attP site, pig-attP-1 was isolated by TAIL PCR. It is located in an intergenic region in pig chromosome 1, spanning from 114220087-114220126. Sanger sequencing showed that pEGFP-N1-attP was broken at attB site and joined with pig-attP-1 site. Extracellular EGFP expression was measured by a fluorescence detector, where its expression level was 50 fold higher than that in background fluorescence (13500AU vs. 280AU), implying that pig-attP-1 site is a safe harbor in favor of transgene expression. This study paves a new way for site-specific transgene integration in pig genome. It also provides a new strategy for creating genetically modified pig and animal bioreactor.