Immortalization of Human Precartilaginous Stem Cells by Transfecting SV40Tag

Immortalized human precartilaginous stem cells （1PSCs） 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 huma...

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Bibliographic Details
Published in:Journal of Huazhong University of Science and Technology. Medical sciences 2009-04, Vol.29 (2), p.231-234
Main Author: 王俊芳 方煌 夏仁云 陈安民 程浩
Format: Article
Language:English
Subjects:
Cartilage - cytology
Cell Proliferation
Cell Transformation, Viral
Cells, Cultured
Fetus
Humans
Medicine
Medicine & Public Health
PCR检测
Simian virus 40
Simian virus 40 - genetics
Southern杂交
Stem Cells - cytology
SV40Tag
Transfection
T抗原基因
免疫组织化学
成纤维细胞生长因子受体3
细胞永生化
细胞转染
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Summary:Immortalized human precartilaginous stem cells （1PSCs） 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 Southem 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.
ISSN:1672-0733
1993-1352
DOI:10.1007/s11596-009-0219-9