含稳定整合pMCLacI/Neo质粒的NIH3T3细胞体外突变研究模型的建立  

Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro

作　　者：卢洋[1] 黎怀星[1] 傅继梁[1] 

机构地区：[1]第二军医大学医学遗传学教研室,上海200433

出　　处：《中华医学遗传学杂志》2000年第2期125-128,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金!(39570400)

摘　　要：目的　建立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突变机理的实验模型。方法　以脂质体转染法将线性化的 p MCL ac I/Neo质粒导入 NIH3T3细胞 ,用 G418筛选 ,选择一个药物抗性细胞克隆进行扩增 ,用基因组 Southern杂交 ,RT- PCR及 RT- PCR Southern杂交进行分子鉴定。结果　 (1)在此细胞克隆的基因组中整合有 p MCL ac I/Neo质粒 ;(2 )该质粒上的两个 lac I靶基因中 ,有一个在 NIH3T3细胞内处于转录表达状态 ;(3)可以通过酶切环化的方法从阳性细胞克隆的基因组 DNA中回收出结构完整、功能正常的 p MCL ac I/Neo质粒。结论　建立了在基因组中整合有 p MCL ac I/Neo质粒的NIH3T3细胞系 ;两个 lac I靶基因可分别模拟哺乳动物细胞内表达基因和沉默基因的功能状态 ;可以将该细胞系用作研究哺乳动物细胞内表达基因和沉默基因的不同突变机理的实验模型。Objective To establish a suitable model for studying the different mechanisms of mutation between expressed and non expressed genes in mammalian cells. Methods The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome mediated transfection,and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT PCR analyses on its genomic DNAs. Results (1)Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac Ⅰ target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/μg DNA. Conclusion The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non expressed genes in mammalian cells.

关 键 词：基因突变 lacI基因 NIH3T3细胞 质粒 

分 类 号：R-332[医药卫生]