Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells

被引:184
作者
O'Hare, MJ
Bond, J
Clarke, C
Takeuchi, Y
Atherton, AJ
Berry, C
Moody, J
Silver, ARJ
Davies, DC
Alsop, AE
Neville, AM
Jat, PS
机构
[1] Royal Free & Univ Coll Sch Med, Ludwig Inst Canc Res, London W1W 7BS, England
[2] Royal Free & Univ Coll Sch Med, Dept Surg, Ludwig Inst Canc Res Univ Coll London Breast Canc, London W1W 7EJ, England
[3] Royal Free & Univ Coll Sch Med, Windeyer Inst, Wohl Vir Ctr, London W1P 6DB, England
[4] Natl Radiol Protect Board, Didcot OX11 0RQ, Oxon, England
[5] Imperial Canc Res Fund, London WC2A 3PX, England
[6] Univ Cambridge, Dept Pathol, Cambridge CB2 1QP, England
[7] Ludwig Inst Canc Res, London SW1E 5AG, England
[8] UCL, Dept Biochem & Mol Biol, London WC1E 6BT, England
关键词
D O I
10.1073/pnas.98.2.646
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction. however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.
引用
收藏
页码:646 / 651
页数:6
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