Characterization And Expression Analysis Of Goat Fetal Fibroblast Cells Induced With Human Telomerase Reverse Transcriptase

Fetal fibroblast cell(sFFCs)are often used as donor cells for nuclear transfer, due to theyare easy to culture, have high growth velocity and stable chromosome ploidy. Even though,the use of FFCs still need to face the difficult of fast ageing, hard to get and hard to expand.What's more, during genetic modification process, which required Fetal fibroblast cells to becultured in vitro for several passages, cells tended to aging very rapidly and became notappropriate donor cells for nuclear transfer. In order to solve the problem of providing dornercells for nuclear transfer, researchers are working on finding suitable techniques andmethords to obtain fetal fibroblast cells who has the character of stable passage and strongproliferative capacity. Human telomerase reverse transcriptase (hTERT) possessed theactivity of human telomerase and maintaining telomere in dividing cells, therefore, hTERTcan be transfected into somatic cells to extend their lifespan.In this study, we transfected a Xinong Saanen Goat fetal fibroblast cells line withpCI-neo-hTERT vector. Then, We tested several characteristics of transfected cells, includinggrowth curve, expression and activity of hTERT, tumorigenicity and expression of oct-4andnanog. The result showed that hTERT could significantly extend the lifespan of transfectedcells in vitro. hTERT mRNA was expressed in hTERT transfected cells. Moreover, hTERTtransfected cells presented enhanced telomerase activity and longer telomere thanuntransfected cells at the same passage. On the other hand, hTERT transfected cells canmaintain normal karyotype even prolonged culture in vitro. After inoculation of hTERTtransfected cells in nude mouse, none of them developed tumors on the vaccination site.Interestingly, Transfection of hTERT can improve expression of Nanog and Oct4in xinongSaanen goat Fetal fibroblast cells, especially in low-generation after transfection, but withincreasing subculture, this effect gradually weakened.The results indicate that hTERT gene modification improved the activity of telomerase, prolonged life span of cells, had good infect on cell survival and proliferation, meanwhilehelped cells to maintain cell biological characteristics. The cell line can be used as donor cellsfor nuclear transfer.