ELECTROPORATION - APPLICATION TO HUMAN LYMPHOID-CELL LINES FOR STABLE INTRODUCTION OF A TRANSACTIVATOR GENE OF HUMAN T-CELL LEUKEMIA-VIRUS TYPE-I
ELECTROPORATION - APPLICATION TO HUMAN LYMPHOID-CELL LINES FOR STABLE INTRODUCTION OF A TRANSACTIVATOR GENE OF HUMAN T-CELL LEUKEMIA-VIRUS TYPE-I
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DOI：
10.1093/nar/17.4.1589
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发表时间：
1989-02-25
影响因子：
14.9
通讯作者：
HINUMA, Y
中科院分区：
文献类型：
--
作者：
OHTANI, K;NAKAMURA, M;HINUMA, Y
通过电穿孔法将外源DNA稳定导入人淋巴细胞系的条件已被开发。为了将人T细胞白血病病毒I型（HTLV-I）的p40基因稳定地导入人淋巴细胞系Jurkat，将携带neo抗性基因的p40表达质粒pMAXRHneo-1在2500 V的电压和21.7 μ F的电容下转染到Jurkat细胞中，并筛选neo（G418）抗性的稳定转化体。转化体的频率大于每2 × 1个。最初使用的105个细胞。对G418具有抗性的克隆显示具有整合到宿主基因组中的p40基因，并且表达来自引入的质粒的mRNA和蛋白质。通过测试HTLV-1增强子对p40的反式激活作用，也证实了p40在转化的Jurkat细胞中的表达。通过电穿孔人T细胞系HSB-2和TALL-1、人B细胞系Raji、人单核细胞系U937和人红白血病细胞系K562也可重复地获得10-4到10-6的高频率稳定转化。这些结果表明，电穿孔是一种非常有效的方法引入外源DNA到人类淋巴细胞系。
Conditions were developed for stable introduction of foreign DNA into human lymphoid cell lines by electroporation. To introduce stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) into the human lymphoid cell line Jurkat, the p40 expressing plasmid, pMAXRHneo-1, which carries the neo resistant gene, was transfected into Jurkat cells at a voltage of 2500 V and capacitance of 21.7 .mu.F, and stable transformants were screened for neo (G418) resistance. The frequency of transformants was more than one per 2 .times. 105 cells used initially. Clones that were resistant to G418 were shown to have the p40 gene integrated into the host genome and to express mRNA and protein from the introduced plasmid. Expression of p40 in the transformed Jurkat cells was also confirmed by testing the trans-activating effect of HTLV-I enhancer by p40. High frequencies of stable transformations of 10-4 to 10-6 were also reproducibly obtained by electroporation of the human T cell lines HSB-2 and TALL-1, a human B cell line Raji, a human monocytic cell line U937, and a human erythrolleukemia cell line K562. These results demonstrate that electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines.