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电场介导的转化:一个TK+亚克隆的分离与鉴定

Electric field mediated transformation: isolation and characterization of a TK+ subclone.

作者信息

Zerbib D, Amalric F, Teissié J

出版信息

Biochem Biophys Res Commun. 1985 Jun 28;129(3):611-8. doi: 10.1016/0006-291x(85)91935-7.

Abstract

Transformation of mammalian TK- cells by a plasmid carrying the TK gene from Herpes virus simplex 1 (pAGO) was mediated by electroporation. The cells were treated either in suspension or growing in monolayers directly in the petri dish. The yield of transformation was between 8.10(-5) and 2.10(-4) per microgram DNA depending on the experimental conditions. The structure of the integrated DNA was investigated proving the occurrence of a duplication process that affected preferentially the pBR322 part of the pAGO DNA (60 copies per cell). The TK gene that gave the TK+ phenotype to the selected clone was present in less than 6 copies.

摘要

携带单纯疱疹病毒1型胸苷激酶(TK)基因的质粒(pAGO)通过电穿孔介导对哺乳动物TK-细胞进行转化。细胞可以在悬浮状态下处理,也可以直接在培养皿中以单层生长的方式处理。根据实验条件,每微克DNA的转化效率在8×10⁻⁵至2×10⁻⁴之间。对整合DNA的结构进行了研究,结果证明存在一个复制过程,该过程优先影响pAGO DNA的pBR322部分(每个细胞60个拷贝)。赋予所选克隆TK⁺表型的TK基因拷贝数少于6个。

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