Introduction of a viral thymidine kinase gene and the human beta-globin gene into developmentally multipotential mouse teratocarcinoma cells.
Introduction of a viral thymidine kinase gene and the human beta-globin gene into developmentally multipotential mouse teratocarcinoma cells.
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将病毒胸苷激酶基因和人β-珠蛋白基因引入发育多能的小鼠畸胎癌细胞中。
DOI：
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发表时间：
1980
影响因子：
11.1
通讯作者：
B. Mintz
中科院分区：
文献类型：
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作者：
A. Pellicer;E. Wagner;A. El;M. Dewey;A. Reuser;S. Silverstein;R. Axel;B. Mintz
畸胎瘤（TCC）干细胞提供了独特的前景，引入特定的基因到小鼠中，凭借其在体外繁殖和胚胎中的正常分化的双重能力。在这项研究中，我们已经证明，在培养中易于选择的外源基因可以转移到干细胞中并表达。在没有选择压力的情况下，这些细胞在体内肿瘤分化期间长时间保持基因表达。这些细胞还以高频率整合未连锁的非选择性基因。将克隆的单纯疱疹病毒（HSV）胸苷激酶（tk; ATP：胸苷5 ′-磷酸转移酶，EC 2.7.1.21）基因加入tk（-）TCC细胞培养物中，以每4 μ g质粒DNA一个菌落的频率产生tk（+）菌落。这种转化效率，虽然明显低于小鼠L tk（-）细胞，允许许多转化体的分离。通过tk蛋白的特征性电泳迁移率和用特异性抗血清中和tk活性来验证转化表型的HSV来源。此外，印迹杂交试验显示至少有一个完整的病毒tk基因拷贝整合到转化细胞的DNA中。当注射到同基因小鼠中时，这些细胞形成了具有各种分化组织的实体瘤。从印迹杂交比较与他们的细胞系的起源，七个检查的九个肿瘤保持HSV tk基因没有显着的损失或重排。病毒tk酶活性也可以在至少一些肿瘤中得到证实。克隆的人β-珠蛋白基因沿着未连接的HSV tk基因的共转移在10个tk（+）转化体中的2个中成功。因此，确定的基因可以稳定地引入培养物中的TCC细胞中，并以它们被转录和翻译以产生功能蛋白的形式在体内维持。
Teratocarcinoma (TCC) stem cells provide unique prospects for the introduction of specific genes into mice, by virtue of their dual capacity for propagation in vitro and for normal differentiation in embryos. In this study, we have demonstrated that foreign genes amenable to selection in culture can be transferred into the stem cells and expressed. These cells maintain expression of the gene for long periods during differentiation in tumors in vivo in the absence of selective pressure. The cells also integrate an unlinked nonselectable gene at high frequency. Addition of the cloned herpes simplex virus (HSV) thymidine kinase (tk; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene to cultures of tk(-)TCC cells yielded tk(+) colonies at a frequency of one colony per 4 mug of plasmid DNA. This transformation efficiency, although appreciably lower than for mouse L tk(-) cells, permits the isolation of many transformants. The HSV provenance of the transformed phenotype was verified by the characteristic electrophoretic mobility of the tk protein and by neutralization of the tk activity with specific antiserum. Moreover, blot hybridization tests revealed at least one intact copy of the viral tk gene integrated into the DNA of transformed cells. When injected into syngeneic mice, the cells formed solid tumors with various differentiating tissues. From blot hybridization comparisons with their cell lines of origin, seven of nine tumors examined had maintained the HSV tk gene without significant loss or rearrangement. Viral tk enzyme activity could also be demonstrated in at least some of the tumors. Cotransfer of the cloned human beta-globin gene along with the unlinked HSV tk gene was successful in 2 of 10 tk(+) transformants. Thus, defined genes can be stably introduced into TCC cells in culture and maintained in vivo in a form in which they are transcribed and translated to produce a functional protein.