title: Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase.
abstract: Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened.
authors.affiliation: College of Animal Veterinary Medicine, Northwest A&F University, Yangling, Xianyang, Shaanxi, 712100, China. xyy0556@126.com
authors.forename: Ying
authors.initials: Y
authors.lastname: Xie
authors.forename: Xiaoe
authors.initials: X
authors.lastname: Zhao
authors.forename: Hongxiang
authors.initials: H
authors.lastname: Jia
authors.forename: Baohua
authors.initials: B
authors.lastname: Ma
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D046149:Embryo Culture Techniques / Q000662:veterinary; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006041:Goats; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007621:Karyotyping; D051379:Mice; D008819:Mice, Nude; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000502:physiology*; D014162:Transfection / Q000662:veterinary
chemical_list: D017931:DNA Primers; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; C509186:TERT protein, human; D019098:Telomerase