Mayne L V, Priestley A, James M R, Burke J F
Exp Cell Res. 1986 Feb;162(2):530-8. doi: 10.1016/0014-4827(86)90356-3.
Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.
永生细胞系对于遗传和生化研究至关重要。与啮齿动物细胞不同，啮齿动物细胞可自发形成持续生长的培养物，或在感染致癌病毒（如猿猴病毒40（SV40））后形成，而人类细胞无法自发形成连续细胞系，且在致癌病毒感染后仅在极少数情况下形成细胞系。我们使用了一种质粒（pSV3gpt），其包含编码T抗原的SV40早期区域和细菌基因黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（gpt），以实现原代人皮肤成纤维细胞的高效形态转化和永生。将该质粒转染到来自一名正常个体、两名科凯恩综合征患者和一名免疫缺陷患者的原代人皮肤成纤维细胞中，并选择gpt基因，导致细胞形态和生长特性发生改变，具有先前描述的SV40转化细胞的特征。转染后的培养物随后衰老、进入危机，并在每种情况下形成快速生长的培养物。此处描述的高效永生化（四个细胞株中有四个）与先前利用局灶性过度生长的程序形成对比。我们认为，使用与SV40早期区域相关联的显性选择标记增加了建立永生人类细胞系的可能性。
