The Construction Of The Porcine Muscle/Liver Tissue-Specific Expression Vectors And The Study Of Transgenic Mouse For MyoD/BGL1

This study to verify promoter activity of the swine muscle-specific gene CAPN3/liver-specific gene TTR. Then We used pig muscle-specific gene myf6promoter and MyoD CDS construct recombinant, which highly expressed MyoD in porcine muscle specificity; Used liver-specific gene TTR promoter with BGL1CDS construct recombinant,which efficient expressed BGL1in pig liver specificity. Transgenic mice were produced by prokaryotic injection techonlogy, This transgenic model is established for the development of high yielding and low consumption of transgenic pigs.The results are as follows:1) Analysis the activity of the muscle-specific expression gene CAPN3promoterThrough the GeneBank database screen porcine muscle-specific expression gene CAPN3and its’DNA seqence, Taked the2300bp upstream of translation initiation site ATG as the research object, We used the NNPP, DNAman, ConSit analysis to predict the core promoter sequence、different species homology、the highest homology region with transcriptional binding factors, According to the preliminary forecast of the internet,eight missing fragments of fluorescence expression vector were constructed,the constructed vector was transfected in C2C12,3T3, Hepal-6cells. To determine P3(-705bp to0) activity highest, and has a certain degree of tissue specificity.2) Analysis the activity of the liver-specific expression gene TTR promoterThrough the GeneBank database screen porcine liver-specific expression of gene TTR and its’DNA seqence, Taked the2500bp upstream of translation initiation site ATG as the research object, We used the NNPP, analysis to predict the core promoter sequence, There were four Score higher core promoter region, then take the full length of2205bp before Transcription initiation site as promoter, to construct fluorescence expression vector, the constructed vector was transfected in C2C12,3T3, Hepal-6cells. The full-length promoter sequence was determine to have a high activity, The expression levels of fluorescence from the different cells of view, there is a strong tissue-specific of the full length promoter sequence. 3) The construction of muscle-specific expression vector pSV40-MYODEGFP-Nl and verification on cell levelThe construction of muscle-specific expression vector pSV40-MYODEGFP-Nl, We used Eukaryotic expression vector pEGFP-N1as a framework, used1422bp myf6gene’s promoter(keep by our team). We used Hind Ⅲ/Kpn Ⅰ double digestion MyoD CDS and pEGPF-Nl vector and recovered large fragment, connected, get the intermediate vector pMYODEGFP-N1. Then used Sal Ⅰ/Hind Ⅲ double digestion pMYODEGFP-N1and SV40-myf6, recovery of large fragments, connected, get final expression vector pSV40-myf6-MYODEGFP-N1. Identified by PCR, restriction enzyme digestion, sequencing correctly, successfully build the muscle-specific expression of MyoD vector. Then authentication of MyoD and EGFP expression on cell level. Cellular level used real-time fluorescent quantitative PCR verification the expression of MyoD and EGFP, in experimental group the expression of MyoD is8.04times compare with the control group, and the expression of EGFP is6.80times compare with the control group.4) The construction of liver-specific expression vector pTTRS-BGLhis-control and verification on cell levelthe construction of liver-specific expression vector pTTRS-BGLhis-control, We used Eukaryotic expression vector pGL3-control as a framework, At first,PCR amplificated BGL1CDS from the pMD-BGL1vector, before that the end of the BGL1CDS plus6xhis label, We used Hind Ⅲ/Xba Ⅰ double digestion pGL3-control vector and BGL1CDS, and recovered large fragment, connected, get the intermediate vector pGL-BGLhis-control.Then used Xho11Hind Ⅲ double digestion pMyoDEGFP-N1and TTR’s promoter plus signal peptide sequence which have been amplificated, recovery of large fragments, connected, get final expression vector pTTRS-BGLhis-control. Identified by PCR, restriction enzyme digestion, sequencing correctly, successfully construct the liver-specific expression of BGL1vector.Then verification the expression of BGL1on cell level.5) The Preparation and identification of transgenic mice for muscle-specific express exogenous gene We used Ase Ⅰ/Afl Ⅱ double digestion to Linearize the muscle specific expression vector pSV40-myf6-MyoDEGFP-Nl, recovery3.5kb target band, The sequence exists Svenhancer enhancer,myf6gene promoter, MyoD and EGFP fusion protein coding sequence, SV40Poly A sequence of these components,Then microinjection, At last, We get4(1male and3female) positive transgenic mice.