含稳定整合pMCLacI／Neo质粒的NIH3T3细胞体外突变研究？…

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ｐＭＣＬａｃＩ／Ｎｅｏ质粒导入ＮＩＨ３Ｔ３细胞，用Ｇ４１８筛选，造反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杂交进行了分子鉴定。结果 （１）在此细胞克隆的基因组中整合有ｐＭＣＬａｃＩ／Ｎｅｏ质粒；（２）该质粒上的两个ｌａｃＩ靶

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

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/microg 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.