Summary
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.
Similar content being viewed by others
References
Peden KWC, Pipas JM. Simian virus 40 mutants with amino-acid substitutions near the amino-terminus of large T antigen. Virus Genes, 1992,6(2):107–118
Pipas JM, Peden KWC, Nathans D. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol Cell Biol, 1983,3(2):203–213
Chen W, Hahn WC. SV40 early region oncoproteins and human cell transformation. Histol Histopathol, 2003, 18(2):541–550
Porras A, Bennett A, Howe K, et al. A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays. Virol, 1996,70(10):6902–6908
Srinivasan AK, Peden WC, Pipas JM. The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions. Virol, 1989, 63(12):5459–5463
Spence SL, Pipas JM. SV40 large T antigen functions at two distinct steps in viron assembly. Virology, 1994, 204(1):200–209
Robinson D, Hasharoni A, Cohen N, et al. Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells. Clin Orthop, 1999,(367Suppl):163–175
He QY, Li QH, Xu JZ. Immortal and biological characteristics of chondrocytes. Chin J Trauma (Chinese), 2001, 17(1):23–25
Greenwood MJ, Lansdorp PM. Telomeres, telomerase, and hematopoietic stem cell biology. Arch Med Res, 2003, 34(6):489–495
Proctor CJ, Kirkwood TB. Modelling cellular senescence as a result of telomere state. Aging Cell, 2003,2(3): 151–157
Shay JW, Van Der Haegen BA, Ying Y, et al. The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with SV40 large T-antigen. Exp Cell Res, 1993,209(1):45–52
DeCaprio JA. The role of the J domain of SV40 large T in cellular transformation. Biologicals, 1999,27(1):23–28
Christiane K, Yoshihiko A, Ilpo H, et al. Immortalization by large T-antigen of the adult epididymal duct epithelium. Mol Cell Endosc, 2004,216(1):83–94
Cavender JF, Conn A, Epler M, et al. Simian virus 40 large T-antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts. Virol, 1995,69(2):923–934
Davies BR, Steele IA, Edmondson RJ, et al. Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen. Exp Cell Res, 2003,288(2):390–402
Darimont C, Avanti O, Tromvoukis Y, et al. SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype. Cell Growth Differ, 2002,13(2):59–67
Kudo Y, Hiraoka M, Kitagawa S, et al. Establishment of human cementifying fibroma cell lines by transfection with temperature-sensitive simian virus-40 T-antigen gene and hTERT gene. Bone, 2002,30(5):712–717
Delgado JP, Parouchev A, Allain JE, et al. Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer. Onco-gene, 2005,24(4):541–551
Steimberg N, Viengchareun S, Biehlmann F, et al. SV40 Large T antigen expression driven by Col2a1 regulatory sequences immortalizes articular chondrocytes but dose not allow stalibization of type II collagen expression. Exp Cell Res, 1999,249(2):248–259
Goldring MB, Birkhead JR, Suen LF, et al. Interlukin-1beta modulated gene expression in immortalized human chondrocytes. Clin Invest, 1994,94(6):2307–2316
Author information
Authors and Affiliations
Additional information
This project was supported by a grant from the National Natural Science Foundation of China (No. 30650007).
Rights and permissions
About this article
Cite this article
Wang, J., Fang, H., Xia, R. et al. Immortalization of human precartilaginous stem cells by transfecting SV40Tag. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 29, 231–234 (2009). https://doi.org/10.1007/s11596-009-0219-9
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11596-009-0219-9