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真核细胞转染后基于EBV的质粒DNA重排

EBV-based plasmid DNA rearrangements after transfection of eukaryotic cells.

作者信息

Morozova O V, Maksimova T G, Kostenko E V

机构信息

Novosibirsk Institute of Bioorganic Chemistry, Lavrentyev's Prospect 8, Novosibirsk, 630090, Russia.

出版信息

Plasmid. 2000 May;43(3):185-9. doi: 10.1006/plas.1999.1449.

Abstract

The cDNA encoding influenza virus (A/Udorn/307/72 strain) M2 protein was subcloned into the EBV-based vector pREP9. Three continuous kidney cellular lines of different origin were transfected with recombinant plasmid pREP9-M2. One and 5 months after transfection plasmid DNA rearrangements were detected by means of restriction analysis of recovered plasmids and their hybridization with an influenza-virus-specific radioactive probe. Deletions were the most frequent type of pREP9-M2 mutations. PCR with primers corresponding to cellular genome and plasmid DNA followed by Southern blot analysis with the [(32)P]-labeled M2-fragment allowed host DNA rearrangements to be revealed in transfected cells.

摘要

编码流感病毒(A/Udorn/307/72株)M2蛋白的cDNA被亚克隆到基于EBV的载体pREP9中。用重组质粒pREP9-M2转染了三种不同来源的连续肾细胞系。转染后1个月和5个月,通过对回收质粒的限制性分析及其与流感病毒特异性放射性探针的杂交检测质粒DNA重排。缺失是pREP9-M2突变最常见的类型。用对应于细胞基因组和质粒DNA的引物进行PCR,随后用[(32)P]标记的M2片段进行Southern印迹分析,可揭示转染细胞中的宿主DNA重排。

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