MicroRNA-7基因敲减小鼠模型的鉴定

发布时间：2018-11-27 13:29

【摘要】：目的鉴定miR-7基因敲减小鼠模型,为进一步研究miR-7的生物学功能奠定基础。方法常规剪取7只miR-7基因敲减(miR-7 knock down,miR-7KD)小鼠的脚趾和尾巴,分别用来提取基因组DNA和总RNA;剪取1只WT(wild-type,WT)小鼠的尾巴,提取其总RNA;将提取的两组小鼠的总RNA逆转录成c DNA;分别以基因组DNA和c DNA为模板,用特异性引物PCR技术扩增目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)片段;提取WT和miR-7KD小鼠7个不同组织器官的总RNA,利用Real-time PCR探针法检测各组织器官中miR-7成熟体的表达水平;HE染色观察miR-7KD小鼠心脏、肺脏以及结肠的形态学结构改变。结果 PCR结果显示,模型小鼠的脚趾基因组DNA和尾巴c DNA中均能扩增出载体目的基因EGFP片段;Real-time PCR结果显示,miR-7KD小鼠心脏、肝脏和脾脏等组织器官miR-7成熟体的表达水平较WT小鼠均显著下调(P0.05);HE染色结果显示,miR-7KD小鼠心脏、肺脏以及结肠形态学结构发生了明显的病理性改变。结论成功鉴定了miR-7基因敲减小鼠模型,为后续深入探讨该分子的生物学功能提供重要实验基础。
[Abstract]:Objective to identify the mouse model of miR-7 knockout reduction and lay a foundation for further study on the biological function of miR-7. Methods the toes and tails of 7 miR-7 gene knockout (miR-7 knock down,miR-7KD) mice were routinely cut. The tails of one WT (wild-type,WT) mouse were extracted from genomic DNA and total RNA;, respectively, and the total RNA; was extracted. The total RNA of the two groups of mice was reverse transcripted to c DNA; using genomic DNA and c DNA as templates respectively. The target gene enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP) fragment was amplified by specific primer PCR technique. The total RNA, of 7 different tissues and organs of WT and miR-7KD mice were extracted to detect the expression of miR-7 matured bodies by Real-time PCR probe method. The morphologic changes of heart, lung and colon of miR-7KD mice were observed by HE staining. Results PCR results showed that the target gene EGFP fragment could be amplified from the toe genomic DNA and tail c DNA of the model mice. Real-time PCR results showed that the expression of miR-7 maturation in heart, liver and spleen of miR-7KD mice was significantly lower than that in WT mice (P0.05). HE staining showed that the morphological structure of heart, lung and colon of miR-7KD mice had obvious pathological changes. Conclusion the mouse model of miR-7 knockout reduction was successfully identified, which provides an important experimental basis for further study on the biological function of the molecule.
【作者单位】：遵义医学院免疫学教研室暨贵州省免疫学研究生教育创新基地;
【基金】：国家自然科学基金资助项目(NO:81260398) 教育部新世纪优秀人才计划项目(NO:NCET-12-0661) 贵州省国际合作项目(NO:10C315)
【分类号】：R-332;R3416

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