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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。
AIM: MicroRNAs (miRNAs) are small RNAs that play a post-transcriptional regulatory role in gene expression and play an important role in both normal development and disease progression. Among them, miR-9 is a highly conserved member of the sequence that regulates many aspects of neural stem cells in neurodevelopment, including differentiation, migration and the like. However, there are some conflicting and unclear functions of miR-9 discovered in the present study. Therefore, we plan to construct an overexpression vector for miR-9 in order to further study miR-9. Methods: We constructed a pcDNA6.2-GW / miR-9 expression plasmid based on pcDNA6.2-GW / miR by using the common method of molecular cloning. We verified the construction of the plasmid by PCR, restriction enzyme digestion and sequencing Correctness, and whether Hela cells and NIH3T3 cells could overexpress miR-9 small molecule could be verified. Results: We successfully constructed the correct pcDNA6.2-GW / miR-9 expression plasmid, and the plasmid can overexpress miR-9 small molecule both in human Hela cells and mouse-derived NIH3T3 cells . Conclusion: Constructing a universal vector of miR-9 between different species laid the foundation for further study on the function of miR-9.