文章摘要
辛 婧,张日华,杜新丽,刘 云.S100A16基因shRNA真核表达质粒的构建及其干扰效果的初步鉴定[J].南京医科大学学报,2011,(3):323~327
S100A16基因shRNA真核表达质粒的构建及其干扰效果的初步鉴定
Construction of shRNA eukaryotic expression plasmid of S100A16 gene and preliminary identification of its interference efficiency
投稿时间:2010-08-11  
DOI:10.7655
中文关键词: 小鼠前体脂肪细胞  RNA干扰  S100A16基因
英文关键词: 3T3-L1 cells  RNA interference  S100A16 gene
基金项目:国家自然科学基金资助(81070684
作者单位
辛 婧 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
张日华 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
杜新丽 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
刘 云 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
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中文摘要:
      目的:构建S100A16基因RNA干扰(RNAi)的真核表达质粒,转染3T3-L1小鼠前体脂肪细胞,初步鉴定其干扰效果?方法:以小鼠S100A16基因为靶基因,以PLKO.1-sP6-GFP 质粒为载体,根据GenBank数据库提供的S100A16基因核苷酸序列,选择设计2条siRNA干扰序列,构建针对S100A16基因的shRNA真核表达质粒PLKO.1-S100A16-GFP-shRNA1?2,经PCR鉴定和测序分析,确认质粒构建成功后,用脂质体LipofectamineTM 2000将重组质粒瞬时转染小鼠3T3-L1小鼠前体脂肪细胞,在荧光显微镜下观察绿色荧光蛋白表达,计算转染效率,Real-time RT-PCR法检测质粒对S100A16基因的表达抑制效果?结果:构建的shRNA序列经PCR和DNA测序证实与设计完全一致;在荧光显微镜下观察到3T3-L1细胞表达绿色荧光蛋白(GFP),证实重组质粒已转入细胞,转染效率达到90%;Real-time RT-PCR结果显示转入PLKO.1-S100A16-GFP-shRNA2的3T3-L1细胞中S100A16基因被特异抑制,基因表达抑制率达70%以上,与空白对照组?阴性序列对照组的差异具有统计学意义(P均<0.05)?结论:成功构建了靶向S100A16基因的shRNA真核表达质粒PLKO.1-S100A16-GFP-shRNA1和2,并筛选出有效抑制S100A16基因表达的质粒,为进一步研究S100A16基因功能奠定了基础?
英文摘要:
      Objective: To construct the eukaryotic expression plasmid carried mouse S100A16 gene short hairpin RNA(shRNA), and to investigate the silencing effect of shRNA on the S100A16 gene in mouse 3T3-L1 cells. Methods:siRNAs were designed according to the S100A16 cDNA sequence in GenBank,and inserted into plasmid PLKO.1-sP6-GFP. The recombinant plasmids were identified by PCR and sequence analysis, and transfected into 3T3-L1 cells with LipofectamineTM 2000. The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by realtime RT-PCR. Results:The correct sequences of recombinant PLKO.1-S100A16-GFP-shRNAs were confirmed by DNA sequencing. Green fluorescence observed by fluorescence microscope showed that shRNAs had been transfected into 3T3-L1 cells(transfection rate was 90%). And the expression of S100A16 mRNA was effectively down-regulated by PLKO.1-S100A16-GFP-shRNA2(inhibition rate was above 70%). Conclusion:The eukaryotic expression plasmids of S100A16 shRNAs were constructed successfully. The PLKO.1-S100A16-GFP-shRNA2 could down-regulated the expression of S100A16 gene effectively. This is fundamental for the study on the function of S100A16 gene.
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