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张正平 赵勤鹏 黄琳红 王栋琪 许正伟 郭华 昌震 刘团江.MicroRNA-9 过表达载体的构建与鉴定[J].现代生物医学进展英文版,2015,15(26):5033-5037.
MicroRNA-9 过表达载体的构建与鉴定
Construction and Identification of microRNA-9 Overexpression Vector
  
DOI:
中文关键词: microRNA  miR-9  过表达载体  pcDNA6.2-GW/miR 载体
英文关键词: microRNA  miR-9  Overexpression plasmid  pcDNA6.2-GW/miR plasmid
基金项目:西安市红会医院院级科研基金项目(YJ2013013)
Author NameAffiliation
张正平 赵勤鹏 黄琳红 王栋琪 许正伟 郭华 昌震 刘团江 西安交通大学医学院附属红会医院 
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中文摘要:
      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pcDNA6.2-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。
英文摘要:
      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 of miR-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 function of miR-9.
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