Immortalization of human precartilaginous stern cells by transfecting SV40Tag

VernacularTitle:猿肾病毒40介导的人前软骨干细胞永生化的实验研究
Author: Janfang WANG ; Renyun XIA ; Huang FANG ; Anmin CHEN ; Delong YI ; Hao CHENG
Publication Type:Journal Article
Keywords: Stern cells; Simian virus 40; Immortalization
From: Chinese Journal of Orthopaedic Trauma 2009;11(3):251-254
CountryChina
Language:Chinese
Abstract: Objective To establish the strain of immortalized human precartilaginous stem cells (PSCs) which can be a stable cell resource for study of the molecular mechanism of gene targeting on differ-entiation of PSCs. Methods Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs using lipofeetin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Immunohistochemistry, RT-PCR and Southern blot were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. Results The positive colonies were isolated and subcultured, named as immortalized precartilaginous stem cells (IPSCs), which were confirmed as positive to fibrnblast growth factor receptor-3 (FGFR-3). The existence of SV40Tag cDNA was detected by Southern blot and the expression of SV40Tag mRNA and protein by RT-PCR and immunohistochemistry. Conclusion IPSCs strain with SV40Tag can be constructed successfully.