穿梭质粒pSP189快速提取和转染的方法  

RAPID METHODS FOR ISOLATION AND TRANSFECTION OF SHUTTLE VECTOR PSP189

作　　者：谈幸之[1,2] 周宇红[1,2] 李申德 罗雪云[1,2] 崔惠云[1,2] 杨晓洁 

机构地区：[1]卫生部食品卫生监督检验所 [2]中国医学科学院肿瘤研究所

出　　处：《癌变．畸变．突变》1996年第6期369-372,共4页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金;卫生部青年科学基金

摘　　要：本文采用基于ＳＶ４０病毒的新型穿梭质粒ｐＳＰ１８９，作为ＰＺ１８９的衍生质粒其独特性在于ｐＳＰ１８９是一个质粒群（ＰｌａｓｍｉｄＰｏｐｕｌａｔｉｏｎ）每个质粒在其靶基因ｓｕｐＦＴＲＮＡ的３’端都插入一段随机产生的８－ｂｐ的标识序列ＳｉｇｎａｔｕｒｅＳｅｑｕｅｎｃｅ），通过对突变子靶基因ＳｕｐＦ和标识序列同时测序可以鉴别出一个转染细胞瓶中两个靶基因序列改变相同的突变子是独立突变（ＩｎｄｅｐｅｎｄｅｎｔＭｕｔａｔｉｏｎ）还是同胞突变（ＳｉｂｌｉｎｇＭｕｔａｔｉｏｎ）。由于ｐＳＰ１８９的独特性，我们总结出一套简便、快速的质粒ＤＮＡ转化、扩增保存、提取、转染及从哺乳动物细胞中回收纯化的方法，结果表明，用我们的一步法从细菌中提质粒和改良的Ｈｉｒｔ法从细胞中回收质粒基本没有细菌和细胞ＤＮＡ的污染，而且不需超离心即可达到较高的纯度并获得较高的转化率和转染率，为进一步筛选分离突变体并对靶基因进行ＤＮＡ序列分析打下了基础。We applied a new shuttle vector plasmid based on SV40 virus for studying mutagenesis in mammalian cells.As a developing plasmid of PZ189,the advantages of pSP189 is a population of plasmid,each of which contains 8-bp signature sequence on 3'site.This wequence confers a unique identification tag to each plasmid and allows individual members to be identified by a distinctive signature.With the signature sequence,we were able to determine whether identical mutations derived from the same transfection were independent mutations or sibling mutations.On account of these advantages of this new vector system,we conclude a set of simple,rapid and effective methods about plasmid DNA transformation,amplifying, saving,isolation, transfection and recovery from mammalian cells.The results show the plasmid DNA isolated by our first-step method from E.Coli MBM7070 and modified Hirt method from mammalian cells were not contaminated by bacteria and cellular DNA.Without super-centrifugation,We could also get high pruity DNA and acquire high-efficiency transformation and transfection,which made foundations for further screening the mutants and sequencing target gene Supf.

关 键 词：穿梭质粒 独立突变 同胞突变 

分 类 号：R114[医药卫生—卫生毒理学] R363[医药卫生—公共卫生与预防医学]