用人DNA介导的基因转移修复中国仓鼠细胞的HPRT缺陷

 中国仓鼠卵巢细胞(CHO-K1)经N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱变和6-巯基鸟嘌呤(6-TG)选择,得到稳定的次黄嘌呤磷酸核糖转移酶(HPRT)缺陷细胞株,酶活性仅为野生型的6.5％。用磷酸钙共沉淀法和电脉冲法向HPRT-细胞转移人宫颈癌细胞(HeLaS_3)基因组DNA,纠正了CHO细胞的HPRT缺陷。酶活性提高了6.9倍,达到野生型的45％。用Alu序列探针进行分子杂交,证实经过基因转移并连续传代15次以上的受体细胞中含人DNA序列。表明人的有关基因已稳定地整合到CHO细胞的染色体中。

Correction of the HPRT Gene Deficiency in Chinese Hamster Ovary Cells by Human DNA-Madiated Gene Transfer

The HPRT deficiency had been induced with MNNG (N-methyl-N'-nitro N-nitrosoguanidine) in CHO-k1 cells and the cell clones were selected in a culture medium containing 6-thio-guanine (6-TG) .Although the cells were cultured for over 15 generations, the phenotype of HPRT- was stable.By the calcium phosphate coprecipitation and the electropration methods, the HeLa S3 DNA was transfered into HPRT gene deficient CHO cells. The' results indicated that the HPRT deficiency in the recipient cells was partially corrected.The relative activity of HPRT in these cells was estimated by incubation with the tritiated hypoxanthine containing substrate followed by radioautography, paperchroma-tography and liquid scintillation counting.The results showed that the activity of HPRT in deficient cells was much lower than that in wild type cells, and the activity of HPRT in gene transfered cells increased markedly.The Alu DNA probe was also used to hybridized the cell DNA dotted on the nitrocellulose filter. 2 μg of HeLa S3 DNA and 10 μg of transformed cell DNA gave the blot hybridization,but 10μg of defiency cell DNA did not.This indicated that human HeLa S3 DNA had been stably integrated the into the chromosome of transformed cells.