pM CLacⅠ/Neo质粒的构建和基因突变研究的新策略

Construction of pMCLacⅠ/Neo plasmid and a new strategy for studying gene mutation

目的：构建一个用于比较研究哺乳动物细胞内表达基因和沉默基因的不同突变机制的质粒载体。方法和结果：设计并构建一种具两个ｌａｃⅠ突变靶基因的突变研究载体：ｐ Ｍ Ｃ ＬａｃⅠ／ Ｎｅｏ 质粒。在该质粒中，一个ｌａｃⅠ靶基因受 Ｃ Ｍ Ｖ 启动子的驱动而使其在人和哺乳动物细胞内能表达，而另一个ｌａｃⅠ靶基因则不能表达。将所获得的ｐ Ｍ Ｃ ＬａｃⅠ／ Ｎｅｏ 质粒经酶切鉴定后，再进行如下功能鉴定：一是分析两个ｌａｃⅠ靶基因在大肠杆菌细胞内的功能状态，结果显示这两个ｌａｃⅠ基因在 Ｄ Ｈ５α宿主菌内功能正常；二是将其导入 Ｎ Ｉ Ｈ３ Ｔ３ 细胞内，分析两个靶基因是否能模拟哺乳动物细胞内表达基因和沉默基因的功能状态。结果表明其中一个ｌａｃⅠ靶基因在 Ｎ Ｉ Ｈ３ Ｔ３ 细胞中处于转录表达状态。结论：本研究构建了一种新型的突变研究载体，位于该载体上的两个ｌａｃⅠ靶基因能模拟人和哺乳动物细胞内表达基因和沉默基因的功能状态。

Objective: To construct pMCLacⅠ/Neo plasmid as an effective vector for studying the different mechanisms of mutation between expressed and non expressed genes in mammalian cells. Methods and Results: A pMCLacⅠ/Neo plasmid, containing 2 copies of lacⅠ gene as the mutation target genes, was designed and constructed by common molecular cloning techniques. In this plasmid, only one of the lacⅠ genes was under the control of CMV promoter and could be transcribed in human and mammalian cells. After structure identification by restriction analysis, pMCLacⅠ/Neo plasmid was further studied with its function. It was first introduced into E.coli cells to test the function of the 2 lacⅠ target genes. The results indicated that both lacⅠ genes could regulate the expression of lacZα reporter gene in DH5α host cells. Whether the 2 target genes could imitate the functional states of expressed and non expressed genes in mammalian cells was also analyzed. A NIH3T3 cell line containing copies of a stably integrated pMCLacⅠ/Neo plasmid was established through liposome mediated transfection, and the functional states of the 2 lacⅠ target genes in the cell line were analyzed with RT PCR. The results showed that only one of the 2 lacⅠ genes could be transcribed in the NIH3T3 cells. Conclusion: A new plasmid as a vector for studying gene mutation, in which 2 lacⅠ target genes could imitate the functional states of expressed and non expressed genes in human and mammalian cells respectively, is constructed.