Plasmid, phage, and genomic DNA-mediated transfer and expression of prokaryotic and eukaryotic genes in cultured human cells.
Plasmid, phage, and genomic DNA-mediated transfer and expression of prokaryotic and eukaryotic genes in cultured human cells.
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质粒、噬菌体和基因组 DNA 介导的原核和真核基因在培养的人类细胞中的转移和表达。
DOI：
10.1159/000132065
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Tischfield,JA
中科院分区：
文献类型：
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作者：
Srivatsan,ES;Stanbridge,EJ;Saxon,PJ;Stambrook,PJ;Trill,JJ;Tischfield,JA
用基因组DNA和克隆基因转染哺乳动物细胞现在是相对常规的。然而，绝大多数研究都使用啮齿动物细胞作为受体。在这里，我们描述了两种人类细胞系的有效转染，次黄嘌呤鸟嘌呤磷酸核糖基转移酶（HPRT）缺陷的HeLa细胞系D98/AH-2和腺嘌呤磷酸核糖基转移酶（APRT）缺陷的HT1080细胞系HTD 114。用Mulligan和Berg的pSV2-gpt质粒转染D98/AH-2细胞，该质粒含有theE。在HAT培养基中选择绿嘌呤-鸟嘌呤磷酸核糖基转移酶（gpt）基因和gpt +转染体。用(1)基因组仓鼠DNA转染htd114细胞，在5 × 10-7M的瓦巴因中选择抗瓦巴因的转染物；(2)采用仓鼠和小鼠基因组DNA，在AAA培养基中选取Aprt+细胞；(3)质粒含克隆的仓鼠或小鼠APRT基因，选择APRT +细胞；(4)采用含有克隆小鼠APRT基因的噬菌体颗粒，选择APRT +细胞。转染效率范围为0.25至1.5 × 103个每µg DNA，在某些情况下进行二次转染。用印迹杂交法检测受体的外源DNA，用选择性培养基细胞生长和供体DNA特有酶的表达检测外源基因的表达。大部分的转染物表现出稳定的跨基因组表达。
Transfection of mammalian cells with genomic DNA and cloned genes is now relatively routine. However, the vast majority of studies have used rodent cells as recipients. Here we describe efficient transfection of two human cell lines, the hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient HeLa line, D98/AH-2, and the adenine phosphoribosyltransferase (APRT)-deficient HT1080 line, HTD 114. D98/AH-2 cells were transfected with the pSV2-gpt plasmid of Mulligan and Berg, which contains theE.colixanthine-guanine phosphoribosyltransferase (gpt) gene, and Gpt+transfectants were selected in HAT medium. HTD 114 cells were transfected with (1) genomic hamster DNA, and ouabain resistant transfectants were selected in 5 × 10–7M ouabain; (2) with hamster and mouse genomic DNA, and Aprt+cells were selected in AAA medium; (3) with plasmids containing either the cloned hamster or mouse APRT genes, and Aprt+cells were selected; and (4) with phage particles containing a cloned mouse APRT gene, and Aprt+cells were selected. Transfection efficiencies ranged from 0.25 to 1.5 × 103transfectants perµg DNA, and in certain cases secondary transfections were done. Foreign DNA in recipients was detected by blot hybridization, and the expression of foreign genes was detected by cell growth in selective media and the expression of enzymes characteristic of the species of the donor DNA. The majority of transfectants showed stable expression of the transgenome.