转基因猪中外源基因拷贝数和整合位点的研究

Transgene Copy Number and Integration Site Analysis in Transgenic Pig

主要采用了绝对定量PCR和热不均一交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR),检测了体细胞核移植技术生产的绿色荧光蛋白转基因猪中外源基因拷贝数和整合位点,并利用旁侧PCR(Junction PCR)对整合位点进行确定,同时进一步分析了整合位点的纯合性.结果表明,绝对定量PCR可以准确有效地检测外源基因拷贝数,标准曲线为:log2N(拷贝数)=-0.9354△Ct+3.4116(R2=0.9974,P<0.001),两只转基因猪中外源基因拷贝数分别为30.85±1.77和18.87±1.34;TAIL-PCR能成功地克隆转基因猪中外源基因整合位点,得到25条特异性条带,经BLAST比对,共获得TgInS1(1440bp)、TgInS2(1263bp)和TgInS3(1861bp)3个整合位点.以整合位点侧翼序列特异性引物与外源基因特异性引物的组合引发Junction PCR,得到预计大小的特异性片段,确定了整合位点上、下游侧翼序列的准确性.采用整合位点5′上游和3′下游侧翼序列特异性引物与外源基因特异性引物的组合,进行Junction PCR,在两只转基因猪中都得到与野生型猪一致的侧翼序列特异性引物扩增片段,表明我们获得的转基因猪都为整合位点杂合子.初步建立了绝对定量PCR和TAIL-PCR对外源基因拷贝数和整合位点检测的体系,为今后研究外源基因在转基因猪中遗传和表达的稳定性打下了基础.

Transgene copy number and integration site were checked in transgenic pigs produced by somatic cell nuclear transfer （SCNT）, moreover, Junction PCR was employed to confirm the integration site and analyze zygosity. The results showed that： absolute quantitative PCR could calculate transgene copy number efficiently. The parameters of the standard curve was： log2N=-0.935 4△Ct＋3.411 6 （R^2=0.997 4, P 〈 0.001）, and copy number were 30.85 ±1.77, 18.87±1.34, respectively, in two transgenic pigs; transgene integration site was successfully cloned by TAIL-PCR, and 25 bands were obtained. Three integration sites, named TglnS1 （1 440 bp）, TglnS2 （1 263 bp） and TglnS3 （1 861 bp）, were detected by BLAST; Junction PCR combining with integration site and transgene specific primers was performed and specific bands confirmed the integration site; Junction PCR combining with 5＇ and 3＇ integration site and transgene specific primers was performed to analyze integration site zygosity. Bands amplified by 5＇ and 3＇ integration site specific primers just as WT control were obtained to determine the heterozygosity of integration site. Absolute quantitative PCR, and TAIL-PCR were established to check transgene copy number and integration site, and it has laid the foundation to study the inheritance and expression stability oftransgene in transgenic animals.