Effect of Telomerase Reverse Transcriptase Gene (TERT) Modification on Proliferation of Mongolian Sheep (Ovis aries) Oviduct Epithelial Cells In vitro
LIU Yang1,2, HAI Le-Si1,2, LIU Yong-Bin3, LIU Chun-Xia1,2, CAO Jun-Wei1,2,*, ZHANG Yan-Ru1,2,*
1 College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China; 2 Inner Mongolia Autonomous Region Key Laboratory of Biomanufacturing, Hohhot 010018, China; 3 Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences, Hohhot 010031, China
Abstract：Oviduct epithelial cells (OECs) can provide an ideal physiological and biochemical environment to support embryonic development, but their proliferation ability in vitro is weak, which can not meet the needs of experimental research and application. In this study, exogenous telomerase reverse transcriptase gene (TERT) was transferred into Mongolian sheep (Ovis aries) oviduct epithelial cells (sheep OCEs, SOECs), and the proliferation ability of Mongolian sheep oviduct epithelial cells modified by telomerase gene was observed in vitro. Firstly, the SOECs system was established by enzyme digestion, and the recombinant plasmid pCI-neo-TERT was transfected into the primary SOECs by liposome method. The results showed that the expression of TERT gene was detected by qPCR; The growth curve and activity of cytokeratin18 (CK18), a specific marker of epithelial cells, were detected by immunofluorescence. The results showed that TERT gene was successfully introduced into SOECs, and PCR showed that 276 bp TERT gene fragment was expressed in the cells, and the expression of TERT gene was significantly higher than that of the uninfected and transfected pCI-neo empty vector (P<0.001). The transfected cells still retained the biological characteristics of SOECs. The doubling time of P5 TERT-SOECs was 46.61 h, while that of P5 SOECs was 65.03 h. It was obvious that the proliferation rate of TERT-SOECs was significantly higher than that of SOECs. In conclusion, the study successfully established the TERT-SOECs system with strong in vitro proliferation ability, which laid an important foundation for the seed cell supply of germplasm cell bank.
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