Transgene Integration Site And Copy Number Analysis In Ttansgenic Cloned Bovine

Transgenic and cloned animals are defined to clone animals on the basis of transgenic animals, and to produce cloning fauna with specific traits, special function or certain purpose. It is the organic combine of transgenic animals and cloned animals.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）. Transgenic animals have been used to produce important proteins, livestock bioreactor, study copy number, integration site, cultivate the disease-resistant strains, xenotransplantation donor and production of bio-pharmaceutical products. It has shown a broad application prospects.After establishment of transgenic animal line, it is important to detectand integration site and transgene copy number, and it is necessary to determine transgene expression and function. Therefore, we checked transgene integration site and copy number in transgenic bovine. Moreover, Junction PCR was employed to confirm the integration site and analyze zygosity.The results were as follows:1. Integration site was successfully cloned by TAIL-PCR, DWACP-PCR and Junction PCR. The results showed that integration of exogenous genes to bovine chromosome in single one point integration. For integration sites were detected by BLAST.2.The results showed that integration of 0504 exogenous genes to bovine chromosome 24 genomic clones （NW₀03104566.1）. Exogenous gene integration caused about 238bp nucleotide in chromosome missing, about 3103bp nucleotide in exogenous gene 3′end missing and AT-rich sequence in intergration site. And two junctions were associated with the consensus sequence for topoisomerase-I cleavage sites. At two genome-transgene junctions, short homologies were found.3. The results showed that integration of Yun exogenous genes to bovine chromosome 16 genomic clones （NW₀03104440.1）, between gene LOC10030 and gene RGL1.Exogenous gene integration caused about 228bp nucleotide in chromosome missing, about 3103bp nucleotide in exogenous gene 3′end missing. Analysis of two genome-transgene junctions showed that‘nibbling’of ends had occurred at some ends to joining. And two junctions were associated with the consensus sequence for topoisomerase-I cleavage sites. At two genome-transgene junctions, short homologies of 1 nt were found.4. Absolute quantitative PCR was used to calculate transgene copy number. The parameters of the standard curve were: Log2N=-0.3075△Ct+0.7421 （R2=0.9996, p<0.001）, and copy numbers were 1.335883, 1.234408, 1.169825, 1.0127. Exogenous gene basic as a single copy integrated into the genome.5. 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.