Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells
Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells
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DOI：
10.1073/pnas.98.2.646
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发表时间：
2001-01-16
影响因子：
11.1
通讯作者：
Jat, PS
中科院分区：
文献类型：
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作者：
O'Hare, MJ;Bond, J;Jat, PS
对于端粒酶活性的单独重组是否足以使不同类型的人类体细胞长生不老，或者是否还需要其他“长生不老”基因编码的额外活性，报告存在分歧。本研究表明，仅异位表达人端粒酶催化亚基（hTERT）或猴病毒40大肿瘤抗原的温度敏感突变体（U19tsA58）都不足以使新鲜分离的正常成人乳腺成纤维细胞和内皮细胞永活。然而，两种基因的结合导致了永生细胞系的有效产生，而不管它们是按什么顺序引入的，也不管它们是在培养物正常增殖寿命的早期还是晚期引入的。转导的顺序和时间。然而，确实影响了基因组的稳定性。核型分析表明，两种转基因均在传代早期引入，hTERT首先产生二倍体细胞系。温移实验表明，永生化状态的维持依赖于功能性U19tsA58大肿瘤抗原的持续表达，单独使用hTERT无法维持U19tsA58大肿瘤抗原在非允许温度下的生长。这种条件二倍体细胞系可为细胞工程、永生化和体外转化的研究提供有用的资源。
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.