Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination
Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination
复制标题
DOI：
10.1073/pnas.93.17.8971
复制
发表时间：
1996-08-20
影响因子：
11.1
通讯作者：
Leboulch, P
中科院分区：
文献类型：
--
作者：
Westerman, KA;Leboulch, P
通过逆转录病毒介导的癌基因转移设计了原代细胞可逆永生化的程序，该癌基因随后可以通过位点特异性重组切除。本研究的重点是永生化的早期阶段：细胞群体的增殖和寿命延长的整体诱导。在该系统中Cre/LoxP和FLP/FRT重组的比较分析表明，只有Cre/LoxP在原代细胞中有效地运作。在对细胞进行差异选择后，获得了癌基因被永久切除的纯细胞群。细胞恢复到其永生化前的状态，如生长特性和p53水平的变化所示，它们的命运符合复制性细胞衰老的端粒假说。通过允许暂时和控制的原代细胞群体的扩增而不保留转移的癌基因，这种策略可以促进对外源性生长因子无反应的细胞的基因治疗操作，并通过体细胞中的同源重组实现实用的基因靶向。逆转录病毒转移和位点特异性重组的组合也应该将基因表达研究扩展到以前无法进行实验的情况。
A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation and life span extension of cell populations. Comparative analysis of Cre/LoxP and FLP/FRT recombination in this system indicated that only Cre/LoxP operates efficiently in primary cells. Pure populations of cells in which the oncogene is permanently excised were obtained, following differential selection of the cells, Cells reverted to their preimmortalized state, as indicated by changes in growth characteristics and p53 levels, and their fate conformed to the telomere hypothesis of replicative cell senescence. By permitting temporary and controlled expansion of primary cell populations without retaining the transferred oncogene, this strategy may facilitate gene therapy manipulations of cells unresponsive to exogenous growth factors and make practical gene targeting by homologous recombination in somatic cells, The combination of retroviral transfer and site-specific recombination should also extend gene expression studies to situations previously inaccessible to experimentation.