Ziv Y, Jaspers N G, Etkin S, Danieli T, Trakhtenbrot L, Amiel A, Ravia Y, Shiloh Y
Department of Human Genetics, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
Cancer Res. 1989 May 1;49(9):2495-501.
Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.
共济失调毛细血管扩张症（A-T）是一种多系统遗传性疾病，其特征为神经退行性变、免疫缺陷、极易患癌、染色体不稳定和放射敏感性。A-T在许多种族群体中都有发现，并且在基因上具有异质性：到目前为止，在A-T中已鉴定出四个互补组。分离A-T基因的尝试部分基于基因转移实验，使用通过SV40转化获得的永久性A-T成纤维细胞系。使用SV40使A-T原代二倍体成纤维细胞“永生化”很困难，这可能是因为这些细胞的染色体不稳定。目前可用的永久性A-T成纤维细胞系数量很少，而且并非所有细胞系都已被归入特定的互补组。通过X射线诱导的DNA合成抑制试验，我们已将A-T菌株AT22IJE归入互补组AB。使用起源缺陷型SV40转染这些细胞，发现一个在危机中存活下来的转化体（AT22IJE-T）具有以这种方式获得的永久性细胞系的典型特征。“凝胶内复性”分析未显示AT22IJE-T中有任何高度的DNA扩增。细胞遗传学分析表明新细胞系中存在相当程度的染色体不稳定，并且这些细胞条件培养基中也含有亲代菌株特有的致断裂活性。“细胞A-T表型”的其他参数在永生化细胞中也得以保留：对X射线和新制癌菌素的致死效应高度敏感，以及“放射抗性”DNA合成。然而，AT22IJE-T对两种DNA损伤剂的敏感性不如亲代细胞明显。通过三种不同的转染方法将质粒pRSVneo导入细胞，测试了细胞摄取外源DNA的能力。使用Chen和Okayama最近发表的方案（《分子细胞生物学》，7: 2745 - 2752，1987），获得了令人满意的G418抗性转染子频率（0.66%），发现该方案优于传统的磷酸钙转染方法和基于聚凝胺的方法。
