miR-137过表达慢病毒的包装和鉴定

doi:10.13481/j.1671-587x.20170406

Abstract

Abstract: Objective: To construct lentiviral vector which can overexpression miR-137and produce lentivirus by lentivirus packaging system, and to explore its infection efficiency and expression in HEK293T cells.Methods: miR-137 sequence was chemically synthesized and cloned into lentiviral vector GV209, and the recombinant plasmid containing human miR-137 was obtained and identified. Then miR-137 recombinant plasmid together with two helper plasmids were transfected into HEK293T cells using Lipofectamine 2000.After the HEK293T cells were infected in multiplicity of infection(MOI) 40 for 48 h, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the expression level of miR-137 was detected by fluorescence quantitative PCR.Results: The sequencing results showed that the inserted gene sequence was completely consistent with the published human miR-137 gene sequence in GenBank. The GFP was observed in the HEK293T cells infected with miR-137 overexpression lentivirus under fluorescence microscope. The fluorescence quantitative PCR results showed that the expression level of miR-137 in the cells infected with overexpression lentivirus was 12.74 times higher than that in the control cells.Conclusion: The lentivirus containing miR-137 gene is successful packaged, and it could efficiently infect the HEK293T cells.

Key words: miR-137, lentivirus, HEK293T cell, green fluorescent protein

CLC Number:

Q786

LYU Dong, LIANG Chunmei, LI Mingying, YIN Jingwen, LUO Xudong, LIN Juda, MA Guoda. Packaging and identification of miR-137 overexpression lentivirus[J].Journal of Jilin University Medicine Edition, 2017, 43(04): 694-697.

References

[1] Yin J, Lin J, Luo X, et al. miR-137:a new player in schizophrenia[J]. Int J Mol Sci, 2014, 15(2):3262-3271.
[2] Mahmoudi E,Cairns MJ. MiR-137:an important player in neural development and neoplastic transformation[J]. Mol Psychiatry, 2017, 22(1):44-55.
[3] Brecevic L, Rincic M, Krsnikz, et al. Association of new deletion/duplication region at chromosome 1p21 with intellectual disability, severe speech deficit and autism spectrum disorder-like behavior:an all-in approach to solving the DPYD enigma[J]. Transl Neurosci, 2015, 6(1):59-86.
[4] The Schizophrenia Psychiatric Genome-Wide Association Study (GWAS) Consortium. Genome-wide association study identifies five new schizophrenia loci[J]. Nat Genet, 2011, 43(10):969-976.
[5] Kwon E, Wang W, Tsai LH. Validation of schizophrenia-associated genes CSMD1, C10orf26, CACNA1C and TCF4 as miR-137 targets[J]. Mol Psychiatry, 2013, 18(1):11-12.
[6] Wright C, Turner JA, Calhoun VD, et al. Potential impact of miR-137 and its targets in schizophrenia[J]. Front Genet, 2013, 4:58.
[7] Kim AH, Parker EK, Williamson V, et al. Experimental validation of candidate schizophrenia gene ZNF804A as target for hsa-miR-137[J]. Schizophr Res, 2012, 141(1):60-64.
[8] Fu J, Zuo X, Yin J, et al. Association of polymorphisms of the receptor for advanced glycation endproducts gene with schizophrenia in a Han Chinese population[J]. Biomed Res Int, 2017,2017:6379639.
[9] Ma G, Yin J, Fu J, et al. Association of a miRNA-137 polymorphism with schizophrenia in a Southern Chinese Han population[J]. Biomed Res Int, 2014, 2014:751267.
[10] Wright C, Calhoun VD, Ehrlich S, et al. Meta gene set enrichment analyses link miR-137-regulated pathways with schizophrenia risk[J]. Front Genet, 2015, 6:147.
[11] Vallès A, Martens GJ, De Weerd P, et al. MicroRNA-137 regulates a glucocorticoid receptor-dependent signalling network:implications for the etiology of schizophrenia[J]. J Psychiatry Neurosci, 2014, 39(5):312-320.
[12] Smrt RD, Szulwach KE, Pfeiffer RL, et al. MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase mind bomb-1[J]. Stem Cells, 2010, 28(6):1060-1070.
[13] Sun G, Ye P, Murai K, et al. miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells[J]. Nat Commun, 2011, 2:529.
[14] Hill MJ, Donocik JG, Nuamah RA, et al. Transcriptional consequences of schizophrenia candidate miR-137 manipulation in human neural progenitor cells[J]. Schizophr Res, 2014, 153(1-3):225-230.
[15] 黄辰,刘利英,倪磊,等.肿瘤miRNAs调控机制的研究进展与展望[J].西安交通大学学报:医学版,2015,36(4):429-434.

Packaging and identification of miR-137 overexpression lentivirus

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