MicroRNA-9过表达载体的构建与鉴定  

Construction and Identification of microRNA-9 Overexpression Vector

作　　者：张正平[1] 赵勤鹏[1] 黄琳红[1] 王栋琪[1] 许正伟[1] 郭华[1] 昌震[1] 刘团江[1] 

机构地区：[1]西安交通大学医学院附属红会医院,陕西西安710054

出　　处：《现代生物医学进展》2015年第26期5033-5037,共5页Progress in Modern Biomedicine

基　　金：西安市红会医院院级科研基金项目(YJ2013013)

摘　　要：目的:MicroRNA(miRNA)是一类对基因表达起着转录后调控的小分子RNA,在正常发育和疾病发生过程中都发挥着重要的功能。其中,miR-9是一个序列高度保守的成员,在神经发育中调节神经干细胞的多种行为,包括分化、迁移等。但是,目前研究发现的miR-9的功能还存在一些相互矛盾和不清楚的地方,因此,我们拟构建miR-9的过表达载体,以便于对miR-9进一步的仔细研究。方法:我们采用分子克隆的常用方法,以pcDNA6.2-GW/miR为基础,构建出pcDNA6.2-GW/miR-9的表达质粒,分别用PCR、酶切和测序的方法验证质粒构建的正确性,并在Hela细胞和NIH3T3细胞中验证该质粒是否可以过表达miR-9小分子。结果:我们成功构建出正确的pc DNA6.2-GW/miR-9表达质粒,并且该质粒无论在人源的Hela细胞还是在小鼠源性的NIH3T3细胞中都可以过表达miR-9小分子。结论:构建了一个在不同种属间通用的miR-9过载体,为我们进一步研究miR-9的功能奠定了基础。Objective： MicroRNA （miRNA） is a kind of small RNA that post-transcriptionally regulates gene expression and plays an important role in normal development and disease development, miR-9 is a highly conserved miRNA and regulates many behaviors of neural stem cells, including differentiation and migration etc. Some discrepancies and ambiguities, however, still exist about the function of miR-9. Therefore, we aimed to construct the overexpression vector ofmiR-9 to facilitate the detailed research of miR-9. Methods： We first employed molecular clone methods to construct the pcDNA6.2-GW/miR-9 expression plasmid based on a linear plasmid named pcDNA6.2-GW/miR. Then the plasmid was tested by PCR, restriction digestion and sequencing. Last, the expression of miR-9 by pcDNA6.2-GW/miR-9 plasmid was verified by transfecting it into Hela cells and NIH3T3 cells. Results： We contructed right pcDNA6. 2-GW/miR-9 plasmid and that pcDNA6.2-GW/miR-9 plasmid could overexpress miR-9 both in human cell line Hela and in murine cell line NIH3T3. Conclusion： We successfully constructed a miR-9 overexpression vector, which could be used to overexpress miR-9 in several cells of different origins. This research provided a basis for our further detailed analysis of the fimction of miR-9.

关 键 词：MICRO RNA MI R-9 过表达载体 PC DNA6.2-GW/mi R载体 

分 类 号：R-33[医药卫生] Q78[生物学—分子生物学]