Abstract
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 (R2=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 TgInS1 (1 440 bp),TgInS2 (1 263 bp) and TgInS3 (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 of transgene in transgenic animals.
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