Establishment Of Mongolian Sheep Oviduct Epithelial Cells Modified By Telomerase Reverse Transcriptase

Oviduct is an important organ for ovum delivery and storage,sperm capacitation,fertilization,and early embryogenesis.Oviduct epithelial cells support embryogenesis and provide the ideal physiological and biochemical environments in the early development.However,oviduct epithelial cells have weaker proliferation in vitro than other somatic cells,and cannot be stored for long as seed cells in the genetic resources center,neither fulfill other experimental research and applications.In view of this,the paper selected the Mongolian sheep as the research objective to cultivate oviduct epithelial cells by means of enzymatic digestion technique,and TERT was transferred into the oviduct epithelial cells by lipofection.With resistance screening of G418,TERT-SOECs was constructed via TERT modification and related verification were performed.The main achievements are as follows:1.SOECs strain was established.The oviduct tissues were obtained from the healthy Mongolian sheep;oviduct epithelial cells were extracted and purified by means of trypsin digestion technique.The cultivated cells showed the typical cobblestone shape of the epithelial cell,with stable expression of specific marks keratin 18(CK18)of the epithelial cell.Subculture show that the cells remain good growth characteristics after cryopreservation and palinesthesia.2.TERT-SOECs was established via TERT modification.Plasmid p CI-neo-h TERT was transferred into SOECs by means of lipofection and TERT-SOECs were obtained with TERT modification through G418 screening;PCR testing has demonstrated that TERT succeeded in transferring into SOECs and currently can transfer to P15,with stable expression of specific marks keratin 18(CK18)of the epithelial cell.The doubling time of TERT-SOECs with P5 TERT modification via in vitro culture was 46.61 h on average,and that of P3 and P5 SOECs were51.86 h and 65.03 h,respectively.Proliferation in vitro of TERT-SOECs with TERT modification was significantly higher than that before the modification,which fulfill the expected effect.In summary,TERT-SOECs with TERT modification established in this paper show strong proliferation in vitro and remain the specific biological characteristics of the oviduct epithelial cells.It provides an essential foundation to the role of TERT-SOECs as the seed cell in the genetic resources center and the future experiments.