【摘要】：目的鑒定miR-7基因敲減小鼠模型,為進(jìn)一步研究miR-7的生物學(xué)功能奠定基礎(chǔ)。方法常規(guī)剪取7只miR-7基因敲減(miR-7 knock down,miR-7KD)小鼠的腳趾和尾巴,分別用來提取基因組DNA和總RNA;剪取1只WT(wild-type,WT)小鼠的尾巴,提取其總RNA;將提取的兩組小鼠的總RNA逆轉(zhuǎn)錄成c DNA;分別以基因組DNA和c DNA為模板,用特異性引物PCR技術(shù)擴(kuò)增目的基因增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)片段;提取WT和miR-7KD小鼠7個不同組織器官的總RNA,利用Real-time PCR探針法檢測各組織器官中miR-7成熟體的表達(dá)水平;HE染色觀察miR-7KD小鼠心臟、肺臟以及結(jié)腸的形態(tài)學(xué)結(jié)構(gòu)改變。結(jié)果 PCR結(jié)果顯示,模型小鼠的腳趾基因組DNA和尾巴c DNA中均能擴(kuò)增出載體目的基因EGFP片段;Real-time PCR結(jié)果顯示,miR-7KD小鼠心臟、肝臟和脾臟等組織器官miR-7成熟體的表達(dá)水平較WT小鼠均顯著下調(diào)(P0.05);HE染色結(jié)果顯示,miR-7KD小鼠心臟、肺臟以及結(jié)腸形態(tài)學(xué)結(jié)構(gòu)發(fā)生了明顯的病理性改變。結(jié)論成功鑒定了miR-7基因敲減小鼠模型,為后續(xù)深入探討該分子的生物學(xué)功能提供重要實(shí)驗(yàn)基礎(chǔ)。
[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.
【作者單位】： 遵義醫(yī)學(xué)院免疫學(xué)教研室暨貴州省免疫學(xué)研究生教育創(chuàng)新基地;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(NO:81260398) 教育部新世紀(jì)優(yōu)秀人才計(jì)劃項(xiàng)目(NO:NCET-12-0661) 貴州省國際合作項(xiàng)目(NO:10C315)
【分類號】：R-332;R3416

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