Research Of Insertion Site Effect Of Exogenous Gene In Transgenic Mouse By Swine MyoD/PPAR-γ2

The expression of the exogenous gene in transgenic animals was affected by the promoter of the expression vector, and also was affected by the insertion site effect that caused when the exogenous gene inserted into the genome. Our laboratory had gotten could be stable genetic MyoD and PPAR-γ2 transgenic(TG) mice by pronucleus microinection previously. In order to study the influenced factors that affected the expression of the exogenous gene in transgenic mice, We established pedigree of the F3 generation MyoD and PPAR-γ2 transgenic mice that our laboratory reserved by pure breeding, and detected the expression of the exogenous gene on DNA, RNA and protein level in the transgenic mice. And we confirmed the locus of the exogenous gene MyoD and PPAR-γ2 by thermal asymmetric interlaced PCR(TAIL-PCR) in the transgenic mouse, determined the integration sites of the exogenous gene and then analyzed the insertion site effect of the exogenous gene, to lay a theoretical foundation for the future application in the production practice.The main research results are as follows:1. We measured the triglyceride content and the protein expression level of PPAR-γ in the hind leg muscle tissue of the PPAR-γ2 transgenic mice and wild-type mice, and the results showed that the triglyceride level in muscle tissue of the PPAR-γ2 transgenic mice was significantly higher than those of wild-type mic(p < 0.05), and the expression of PPAR-γ protein in the PPAR-γ2 transgenic mice was higher than those in wild-type mice;2. We detected the expression of exogenous gene MyoD in each different tissue of the MyoD transgenic mice by RT-qPCR, the results indicated that the expression of MyoD gene in muscle tissue was significantly higher than other tissue, and the expression of the MyoD protein in hind leg muscle in the MyoD transgenic mice was significantly higher than those in wild-type mice;3. We established pedigree of the two kinds of transgenic mouse by pure breeding, and we got homozygous positive transgenic mouse by pure breeding from generation to generation and preliminary detection;4. We confirmed the locus of the exogenous gene MyoD and PPAR-γ2 in the transgenic mouse by thermal asymmetric interlaced PCR(TAIL-PCR), the results showed that the exogenous gene PPAR-γ2 inserted into chromosome 9 RP23-366E1 in the PPAR-γ2 transgenic mice; and the exogenous gene PPAR-γ2 existed series insertion; and the exogenous gene MyoD existed series insertion; and we analyzed the 2000 bp nucleotide sequences of the both flanks adjoined to the integration site of the exogenous gene PPAR-γ2 by ORF Finder, and found ORFs in both flanks adjoined to the integration site of the exogenous gene PPAR-γ2; and we used Promoter 2.0 and Promoter Scan to predict the existence of promoter in the 2000 bp nucleotide sequences upper streamed the integration site of the exogenous gene PPAR-γ2, the results showed there was endogenous promoter in the 2000 bp nucleotide sequences;5. We used the absolute quantitative PCR to measure the copy number of the exogenous gene PPAR-γ2 and MyoD in the transgenic mice, the data indicated that exogenous gene could integrate into the transgenic mouse genome and the copy number in the transgenic mouse genome increased from generation to generation until getting positive homozygous transgenic mice;6. We predicted the existence of CpG island in the exogenous gene PPAR-γ2 and MyoD via online software, the results showed that the expression vector pGL3-Myoz1-PPAR-γ2his-Basic of the exogenous gene PPAR-γ2 did not have CpG island; and the expression vector pSV40-Myf6-MyoDEGFP-N1 of the exogenous gene MyoD had 3 CpG islands and one of them located at the promoter region of the expression vector. Then we analyzed the methylation situation of the exogenous gene MyoD in the MyoD transgenic mice by bisulfite PCR-SSCP, and found the methylation level of the CpG island in the Myf6 promoter region in the expression vector of the exogenous gene MyoD was so high.In conclusion, in the PPAR-γ2 transgenic mice, the expression of the exogenous gene PPAR-γ2 was significantly higher; and the exogenous gene PPAR-γ2 located on chromosome 9, the expression vector of the exogenous gene PPAR-γ2 did not have CpG island; and the exogenous gene PPAR-γ2 was multicopy insertion. In the MyoD transgenic mice, the expression of the exogenous gene MyoD in the transgenic mice was higher than in wild-type mice, and the expression of MyoD gene in muscle tissue was significantly higher than other tissue in the transgenic mice, and there was 3 CpG islands around the expression vector of the exogenous gene MyoD, and one of them located at the Myf6 promoter region, methylation detection showed methylation level of the CpG island lacted at the Myf6 promoter region of the expression vector of the exogenous gene MyoD was so high, and the exogenous gene MyoD was multicopy insertion.