Construct Muscular Specific Expcific Expression Vector Of Piggybac Transpon Mediated Bovine A-FABP And Screen The Transgenic Cell Lines

Beef were abundant in protein, and its amino acid component was more similar to people than pork. So, it was popular among consumers as the second largest meat consumption in China. Recently, with the improvement of people’s living standards, great progress has been made on Chinese Yellow Cattle. But the beef with excellent quality were low in yield, and the content of intramuscular fat was also low. So far, molecular breeding, especially transgenic breeding, has been paid more attention. Through transgenic method, how to culture excellent beef cattle breed with good production performance and improve the content of intramuscular fat had been a hot point in present study. Fat acid binging protein had a great relationship with the synthesis and decomposition of triglyceride. Through PiggyBack transposon-mediated specific expression of fat acid binding protein gene in muscle, the content of intramuscular fat and the quality of beef can be improved.In this study, the PiggyBac transposon-mediated vector with specific expression in muscle had been constructed; its expression in bovine myoblast had been inspected and verified; the integration cites of PiggyBac transposon in bovine genome had also been analyzed; the cell line transfected with A-FABP was screened; the advantages and disadvantages of PiggyBack transposon in producing transgenic cattle were evaluated; basis had been made for culturing transgenic cattle with abundant intramuscular fat. The results of this study were as follows:1. Bovine A-FABP gene had been cloned. Sequencing analysis demonstrated that there was no mutation in this gene. So, it can be used for the following experiment. Bovine a-actin was a kind of skeletal muscle specific promoter. Its promoter sequence was cloned. Sequencing analysis showed that there were two mutations in the cloned sequence. Further analysis suggested that the two mutations were not in the region of transcription factor binding site of a-actin, which suggested that the binding of a-actin to transposon factor would not be affected by these two mutations. Cloned the green fluorescent protein EGFP gene expression cassette and Neomycin resistance gene expression cassette, what had been remolded. EGFP gene expression cassette and Neomycin resistance gene sequence frame anchored through LoxP sequence was obtained LoxP-EGFP-NEO-LoxP sequence. In earlier transgenic studies, this sequence can be used for verifying the kinds of cell and screening cell lines. Later stage, it can be cut off by recombinase Cre. The PiggyBack transposon-mediated vector with specific expression of A-FABP in muscle had been constructed. In this study, EGFP was used as reporter gene; resistant gene Neomycin was used for screening; EGFP and Neomycin were anchored by LoxP sequence; the regulatory sequence of α-actin was used as muscular specific promoter; A-FABP was the target gene. The results of enzyme digestion and sequencing suggested that this vector was constructed successfully.2. Results suggested that the ability of PiggyBack binary system mediated expression of exogenous gene was significantly higher than PiggyBac transposon vector and pJFI-EGFP-NEO plasmid vector. As binary system, the cotransfection of pPB-AFABP and pCAG-PBase was carried on in bovine myoblast. The results of RT-PCR showed that the expression level of A-FABP was higher in transfected cells than the cells without transfection. It was suggested that α-actin promoter had high transcriptional activity. Compared transcriptional activity of α-actin promoter and CMV promoter, results suggested that the activity ofa-actin promoter was one sixth of CMV promoter.3. With different transfection concentration in fibroblast, the activity of pPB-AFABP and pCAG-PBase was inspected and verified., the number of cell colonies was calculated. Results suggested that pPB-AFABP and pCAG-PBase had relatively higher activity with the ratio5:1to1:2. The highest activity was shown at the ratio2:1. The cotransfection of pPB-AFABP and pCAG-PBase with the ratio2:1was carried on in cattle fibroblast. Stable transgenic cell line was obtained by G418screening.4. The integration site of PiggyBac transposon on cattle genome was detected through Genome Walking Kit. The results showed that, PiggyBac transposon integration sites in bovine genome tend to between genes and gene intron, and the tendency to be inserted in the AT-rich nucleotide sequence.