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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 an experimental model for the comparative study of the mutational mechanism of expressed genes and silent genes in mammalian cells. Methods The plasmid p MCL ac I / Neo was transfected into NIH3T3 cells by lipofectamine. The cells were screened by G418 and selected a drug-resistant cell clone for amplification. Genomic Southern hybridization, RT-PCR and RT-PCR Southern hybridization for molecular identification. Results (1) The p MCL ac I / Neo plasmid was integrated into the genome of this cell clone; (2) one of the two lac I target genes on this plasmid was transcriptionally expressed in NIH3T3 cells; (3) Plasmid p MCL ac I / Neo, which is structurally intact and functionally normal, can be recovered from the genomic DNA of positive cell clones by enzymatic cleavage. Conclusions A NIH3T3 cell line integrating the p MCL ac I / Neo plasmid was established in the genome. The two lac I target genes were able to mimic the functional status of the expressed and silenced genes in mammalian cells, respectively. The cell line can be used as a study Experimental models of different mutations in mammalian expression genes and silencing genes.