Immortalization of human precartilaginous stem cells by transfecting SV40Tag.
Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.
Wang
Junfang
J
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. wjf09@yahoo.cn
Fang
Huang
H
Xia
Renyun
R
Chen
Anming
A
Cheng
Hao
H
D002356:Cartilage / Q000166:cytology*; D049109:Cell Proliferation; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D005333:Fetus; D006801:Humans; D013539:Simian virus 40 / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection