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目的研究神经干细胞特异性调控启动子。方法 PCR扩增出小鼠nestin基因启动子全序列(4 000 bp)、核心序列(400 bp)和内含子-2;以pcDNA3.1为模板,构建6种重组质粒,分别用CMV、CMV+内含子-2、nestin基因启动子全序列、nestin基因启动子全序列+内含子-2、nestin基因启动子核心序列、nestin基因启动子核心序列+内含子-2作为启动子调控报告基因EGFP表达;6种重组质粒分别转染nestin~+、nesti~-细胞,荧光显微镜下观察转染后细胞内EGFP表达,同时采用流式细胞仪法测定表达EGFP细胞表达率。结果 nestin基因启动子全序列及核心序列都能非特异性调控EGFP基因在细胞内表达,并且具有较强调控能力,与内含子-2融合后,只能特异性调控EGFP基因在nestin~+细胞表达,而CMV启动子与内含子-2序列融合只具有非特异性调控能力。结论 nestin基因启动子全序列与内含子-2协同调控外源基因在nestin~+细胞特异性表达。
Objective To study the specific regulation of neural stem cells promoter. Methods The full length (4 000 bp) and core sequence (400 bp) of mouse nestin gene and intron 2 were amplified by PCR. Six recombinant plasmids were constructed by using pcDNA3.1 as template, Intron-2, nestin gene promoter full sequence, nestin gene promoter full sequence + intron-2, nestin gene promoter core sequence, nestin gene promoter core sequence + intron-2 as a promoter regulatory report EGFP was transfected into nestin ~ +, nesti ~ 6 cells respectively. The expression of EGFP in transfected cells was observed by fluorescence microscopy. The expression of EGFP was detected by flow cytometry. Results The full - length and core sequence of the nestin promoter both could regulate the expression of EGFP gene in the cell nonspecifically, and had strong regulation ability. After fusion with intron - 2, EGFP gene could specifically regulate the expression of EGFP gene in nestin ~ + cells While CMV promoter and intron-2 sequence fusion only have nonspecific regulatory ability. Conclusion The complete sequence of nestin gene promoter and intron - 2 cooperate to regulate the expression of exogenous genes in nestin ~ + cells.