Copy Number And Integration Site Of Transgene In Transgenic Pig

Transgenic animals have been used to study gene function, produce important proteins, xenotransplantation donor and generate models for the study of human diseases. Recent progress in animal cloning has provided an attractive alternative to improve transgenic efficiency, through the combination of transfection and somatic cell nuclear transfer (SCNT). Now many kinds of transgenic animals have been successfully produced by SCNT, such as mice, cattle, sheep and pig, and we also produced eGFP transgenic pigs by SCNT in 2008. After establishment of transgenic animal line, it is important to detect transgene copy number and integration site, and it is necessary to determine transgene expression and function. Therefore, we checked transgene copy number and integration site in founder transgenic pigs and its offspring. Moreover, Junction PCR was emplyed to confirm the integration site and analyze zygosity.The results were as follows:1. PCR and Southern Blot were emplyed to identify the founder transgenic pigs and its offspring, and two founder transgenic pigs and seven offspring were verified.2. Absolute quantitative PCR was used to calculate transgene copy number. The parameters of the standard curve was: log2N=-0.9354△Ct+3.4116 (R2=0.9974, p<0.001), and copy number were 30.489±1.7696, 18.8676±1.3383, 7.0141±0.3441, 6.859±0.5157, 9.8897±1.0931, 9.1881±1.2008, 3.6708±0.5886, 5.7114±0.8494 and 9.5404±0.8438, respectively, in founder transgenic pigs and seven offspring.3. Integration site was successfully cloned by TAIL-PCR and LM-PCR, and 25 bands and 12 bands were obtained respectively.4. Three integration sites, named TgInS1 (1440bp), TgInS2 (1263bp) and TgInS3 (1861bp), were detected by BLAST. TgInS1 in K25-2 was in a L1M LINE element, and TgInS2 and TgInS3 were obtained a hit with significant match in the pig EST database.5. Junction PCR combining with integration site and transgene specific primers was performed and specific bands confirmed the integration site.6. 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. The result showed that absolute quantitative PCR, TAIL-PCR and LM-PCR were established to check the transgene copy number and integration site, and it has laid the foundation to study the inheritance and expression stability of transgene in transgenic animals.