miR375表达质粒构建及表达研究

Study of the miR375-expression plasmid construction

目的寻找一种高效、简便的构建表达miRNA质粒的方法。方法该研究用PCR方法直接从小鼠gDNA扩增了包含miRNA375(miR375)前体序列在内的一个126bp片段以构建质粒pAAV-miR375。该研究用pAAV-miR375转染Hela细胞48h后,用定量RT-PCR方法和Northernblot检测Hela细胞中miR375的表达。结果转染了pAAV-miR375的Hela细胞中有miR375的表达,而在转染了空质粒pAAV-MCS和未转染的Hela细胞中没有检测到miR375的表达。结论该研究所构建的pAAV-miR375质粒能够表达miR-NA375。作者介绍的这一种构建表达miRNA质粒的方法比以往的方法更快速、高效、简便和保证序列的正确性。

【Objective】 To find a more effective and convenient method to construct miRNA-expression plasmid.【Methods】 We directly cloned a 126-bp fragment including miR375 precursor from the mouse gDNA to construct pAAV-miR375 plasmid. The plasmid was transfected into the Hela cells and the expression of miR375 was analyzed by Real-time PCR and Northern blot analysis. 【Result】The miR375 expression was detected in the Hela cells transfected with pAAV-miR375 while was not detected in the Hela cells transfected with control plasmid or untransfected.【Conclusion】 The method we used to construct plasmid is effective and convenient.