Preliminary Research On Construction Of Transgenic Pig Cell Line Model With PERV Targeting Vector

Pig, as the most important edible animal, some aspects of its physiological structures are similar with human’s, so it also becomes an important animal model in medical research and significant organ donor in xenogeneic transplantation. But there is still no efficient way to construct pig’s animal models and cell models. In the common transgenic methods, the foreign genes usually insert in a random site of the genome, which may disrupt some important genes in the host or cause developmental defects. It is significant for cultivation of transgenic pigs and construction of animal models to establish a new transgenic method with high efficiency.Pig’s genome contains many copies of porcine endogenous retrovirus(PERV). In general, these provirus genomes have gene defects, and no influences on the functional genes of the host. They will replicate as long with the chromosomes of host. In this research, a gene targeting vector was constructed based on the porcine endogenous retrovirus(PERV) with the LTRs in PERV used as homologous sequences, the foreign gene was integrated on the PERV sites in pig’s genome through homologous recombination. After the stable integration was selected, homologous recombination sites were detected through PCR, the results showed that foreign gene did integrate into genome by homologous recombination. This method not only increases the efficiency of integration of foreign genes, but also enhances the expression of foreign genes without interrupting functional genes of the host. Works in this research including:1. Construction of PERV targeting vector The 5’LTR -ψ in the pPM-1-GFP-21 was cut off by double digestion with Kpn Ⅰ and Xho Ⅰ. The CMVie promoter was obtained from vector pEGFP-N3 by PCR, then was added at the down stream of 5’LTR,5’LTR -ψ was replaced by 5’LTR-CMVie. After verified by digestion with restriction enzymes, a new vector was built named as pPCM1. Then the HSVtk gene was amplified from vector pORF9-HSVltk by PCR, and added at the down stream of 3’LTR of pPCM1 through digestion with restriction enzyme, ligation and PCR. After checking the position and direction of HSVtk gene, the target vector pPCM1-tk was constructed. pPCM1-tk and pPM-1-GFP-21 separately transfected 293T, the result showed that pPCM1-tk had higher efficiency, which means CMVie promoter enhanced effect of the vector.2. Selection of transfection reagents and cells Different cells were transfected with different reagents to make sure the best reagent and cell would be used in this research.5 different kinds of pig cells were chose in my research--ST, SK-6, PK15, IBRS2, CPK. The first reagent used in the research was LipofectamineTM 2000, and results showed that the effect in ST and SK-6 was a little better. Other reagents were EntransterTM-D and X-tremeGENE HP. The results showed that the X-tremeGENE HP was better for its low toxicity and high efficiency, and should be chose for further experiments.3. Transfection and isolation of cells The ST and SK-6 were cultivated with different concentration of G418 and GANC for different days to find out the best condition to isolate the cells in which homologous recombination happened. The results showed that, ST should be cultivated in G418 600ug/ml for 10 days and GANC 1000ug/ml for 2 days, and.SK-6 should be cultivated in G418 400ug/ml for 10 days and GANC 500ug/ml for 2 days. After isolation of the target cell with condition above mentioned, their genome was extracted.4. Detection of homologous recombination 4 different sites were chose to detect whether and where the homologous recombination had happened, the results of PCR showed that 3 of these sites existed in pig’s genome, and homologous recombination happened in 2 of these sites in SK-6, but not happened in ST. But there are many copies of PERV in the genome of pig, and homologous recombination can happen in each of these copies, so detections on other sites may be positive, more experiments should be conducted.Above all, a gene targeting vector was constructed, which was later transfected to ST and SK-6, stable transgenic cell lines which the foreign gene was integrated in the genome through homologous recombination was isolated, the sites homologous recombination may happened were detected by PCR. This research showed feasibility of construction of transgenic cells with PERV gene targeting vector.