Constructions And Detection Of Expression And Co-expression Vectors Of Follistatin And Fat-1 Genes In Bovine

In the present study, the eukaryotic expression vectors of pIRES2-AcGFP1-FST and pFST-IRES2-FAT1 were constructed. The vectors were then transfected into wagyu fetal fibroblast cells respectively and the positive cells were used as donors to create cloned embryos. The cloned blastocysts were then identified by RT-PCR using total RNA as template for the expression of bovine follistatin cDNA and fat-1. The followings are the major results.1. Construction of eukaryotic expression vector pIRES2-AcGFP1-FSTTotal RNA was extracted from bovine ovary by Trizol total RNA Extract Kit, and the whole coding region of bovine follistatin cDNA gene was cloned by using specific primers which have restriction enzyme cutting site. The purified bovine follistatin cDNA was inserted into pMD18-T vector and sequenced. Subclone the correct follistatin cDNA to eukaryotic expression vector pIRES-AcGFP. Double-enzyme cleavage and PCR test showed that FST was successfully cloned in eukaryotic expression vector.2. Construction of eukaryotic expression vector pFST-IRES2-FAT1The whole coding region of fat-1 cDNA gene was cloned by using specific primers which have restriction enzyme cutting site. The purified fat-1 cDNA was inserted into pMD18-T vector and sequenced. Subclone the correct fat-1 cDNA to eukaryotic expression vector pIRES2-AcGFP1-FST, replacing the AcGFP gene by fat-1 cDNA. Double-enzyme cleavage and PCR test showed that fat-1 was successfully cloned in eukaryotic expression vector pIRES2-AcGFP1-FST.3. In vitro culture and gene transfection of wagyu fetal fibroblast cellsWagyu fetal fibroblast cells were successfully isolated by attachment of tissue pieces from a fetus of wagyu. The expression vector pIRES2-AcGFP1-FST and pFST-IRES2-FAT1 were transfected into wagyu fetal fibroblast cells genome by Lipofectamine LTX. The transgenic clones were selected by G418, and the pIRES2-AcGFP1-FST transgenic clones were selected by green fluorescence. The pFST-IRES2-FAT1 transgenic clones were PCR-identified positive.4. Production and identification of the transgenic cloned embryosThe cloned blastocysts were obtained by routine somatic cell nuclear transfer through the process including enucleation, donor cell injection, fusion and embryo culture. Total RNA was extracted from the transgenic embryos by Trizol total RNA Extract Kit and analysed by PCR, which proved the cloned embryos were transgenic positive.