MicroRNA-7基因敲减小鼠模型的鉴定

Identification of murine microRNA-7 knockdown model

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(P<0.05);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。

Objective To identify the microRNA-7 knockdown（ miR-7KD） mice for successive research on its biological function. Methods Genomic DNA and total RNA were extracted from the toes and tails of seven miR-7KD mice. Total RNAs were extracted from the tail of one wild type（ WT） mice and then reverse transcripted into c DNAs. Using genomic DNA and c DNA as template respectively,the objective fragment EGFP was amplified by PCR using specific primer. Total RNAs were extracted from seven kinds of tissues and organs in WT and miR-7KD mice. Then,the relative expression of miR-7 in seven kinds of tissues and organs were detected using Real-time PCR. Moreover,the morphological changes of heart,lung and colon were observed by HE staining. Results The objective fragment EGFP were amplified in Genomic DNA and c DNA from miR-7KD mice.Compared with seven tissues and organs of WT mice,the relative expression of miR-7 in heart,liver,spleen,lung,kidney,thymus and pancreas from miR-7KD mice were significantly decreased（ P〈0. 05）. HE staining showed that the morphological structures of heart,lung and colon from miR-7KD mice were significantly abnormal compared with those from WT mice. Conclusion The miR-7KD mice model has been identified successfully,which may provide a useful animal model for successive investigation on the biology function of miR-7 molecule.