Studies Related To Transfection Of Buffalo Fetal Fibroblasts With Htert Gene And Nuclear Transplantation

Fetal fibroblasts are usually used as donor cells for somatic cell nuclear transfer (SCNT), however, their limited passages subculture in vitro and injury problems after genetic modification limit the application in SCNT. Human telomerase reverse transcriptase (hTERT) is the main component of telomerase and plays an important role in the process of maintaining and extending telomeres. The purpose of this study was to construct the eukaryotic expression vector of hTERT gene which was used to infect the buffalo fetal fibroblasts (BFF), so that the lifespan of buffalo fetal fibroblasts subculture in vitro was extended and a cell line which could stand the genic modification and drug selection was obtained to improve the efficiency of transferred genic cloning in animals.Firstly, the retroviral vectors of pMXs-hTERT-NEO-GFP and pMXs-hTERT-IRES-NEO were successfully reconstructed and respectively tranferred into buffalo fetal fibroblasts (BFF) by retrovirus, the BFF were obtained by tissue explant culture. The transgenic cell colonies were selected by combining high pressure selection (800μg/mL G418) with constant selection (200μg/mL G418), and then the cell lines of tranferred hTERT gene were ulteriorly enlarged culture. Secondly, the relative expression levels of hTERT in BFF and the BFF respectively transfected with the vectors of pMXs-hTERT-IRES-NEO (phn-BFF) and pMXs-hTERT-NEO-GFP (phnG-BFF) were detected, and found that the relative expression levels of hTERT between the5th and30th passage of phn-BFF was no significant difference (P>0.05), but both of them were significantly higher than the5th passage of phnG-BFF and the5th passage of BFF (P<0.01), and the5th passage of phnG-BFF was also significantly higher than the5th passage of BFF (P<0.01). And then, the biological characteristics of the30th passage of phn-BFF such as karyotype, cell cycle, the protein and activity of telomerase, soft agar assay and the relative expression levels of p53gene were respectively analysed. Karyotype analysis found that the percentage of diploid chromosome in the30th passage of phn-BFF was85%. The cell cycle analysis demonstrated that the30th passage of phn-BFF was significantly increased on the percentage of mitotic phase (S phase) cells (phn-BFF38.6%>BFF25.5%, P<0.05) and was evidently decreased on the percentage of G0+G1cells (phn-BFF45.4%<BFF63.6%, P<0.05) compared with the5th passage of BFF, and the proliferation index (PI) of phn-BFF Cell was significantly enhanced (52.9%VS33.2%, P<0.05) compared with the5th passage of BFF. Moreover, the phn-BFF which transferred hTERT gene could express and translate bioactive telomerase, and had no neoplastic transformation after detected by soft agar assay. The relative expression levels of p53in the30th passage of phn-BFF was significantly decreased than the30th passage of BFF (P<0.05), but there was no significant diference with the5th passage of BFF (P>0.05).Lastly, the developmental ability of buffalo SCNT embryos respectively from the donor cells of5th,30th passage of BFF and the30th passage of phn-BFF was examined, and the relative expression levels of Cdx2and Cx43gene in SCNT blastocyst were detected by Q-PCR technology. The results showed that the fused rate, cleavage rate and blastocyte development rate of reconstructed embryos from the30th passage of phn-BFF had no significant diference with the5th passage of BFF (P>0.05), but there was significantly higher than that of the30th passage of BFF (P<0.05). Moreover, the Q-PCR analysis found that the relative expression levels of Cdx2in the reconstructed embryos from the30th passage of phn-BFF was significantly increased than that of the30th of BFF (P<0.05), while it was no significant difference with the5th passage of BFF (P>0.05), furthermore, the relative expression levels of Cx43in the reconstructed embryos from the30th passage of phn-BFF was significantly improved than that of the5th and30th passage of BFF (P<0.05).In conclusion, the retroviral vectors of pMXs-hTERT-NEO-GFP and pMXs-hTERT-IRES-NEO are successfully reconstructed and tranferred into buffalo fetal fibroblasts (BFF), and the BFF transferred hTERT gene not only express hTERT gene and translate bioactive telomerase, but also remain normal biological characterristics and extend their lifespan. Moreover, the BFF transferred hTERT gene as donor cell for SCNT may direct embryonic development more effectively than BFF after long subculture in vitro.