目的 构建人miR-1908慢病毒表达载体，包被病毒并检测其在人脂肪前体细胞中过表达效果。方法 以人脂肪细胞基因组DNA为模板，PCR高保真扩增包含miR-1908区域上下游各100bp左右的基因组DNA，亚克隆入慢病毒表达载体，与包装质粒共转染HEK-293T细胞，包装病毒，收取病毒上清，纯化后感染人脂肪前体细胞，36 h开始观察荧光标记，72 h收取细胞，抽提RNA，Realtime PCR检测慢病毒感染下miR-1908的相应表达量。结果 成功构建人miR-1908慢病毒表达载体，包装获得的病毒感染人脂肪前体细胞的效率可达到70%以上，miR-1908过表达水平可接近2倍。结论 本研究成功构建了人miR-1908慢病毒过表达载体，包被的慢病毒可以在人脂肪前体细胞中实现过表达效果，为后续功能研究奠定了基础。
Abstract
Objective To construct a recombinant lentiviral vector carrying miR-1908 and validate its infection efficiency in human pre-adipocytes. Methods The minigene fragments of miR-1908 was amplified from genomic DNA of human pre-adipocytes by PCR,then cloned into a lentivirus expression vector.After sequencing validated exactly,the expression plasmid and packaging plasmids were co-transfected into HEK-293T cells to produce lentivirus;The supernatant containing lentiviral particles was used to infect human pre-adipocytes,and its infection efficiency and miR-1908 overexpression level were respectively tested by fluorescent observation and real-time quantitative PCR. Results miR-1908 lentiviral vector was constructed successfully,and infection efficiency in human pre-adipocytes reached over 70%,the expression level increased nearly two folds in infected cells. Conclusion It has been successfully constructed that the lentiviral vector containing miR-1908 with high overexpression in infected human pre-adipocytes,which will be useful to the future studying the function of miR-1908 in human pre-adipocytes
关键词
miR-1908 /
慢病毒 /
人脂肪前体细胞
Key words
miR-1908 /
lentivirus /
human preadipocytes
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参考文献
[1] Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[2] Lai EC.Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation[J].Nat Genet,2002,30(4):363-374.
[3] Ke XS,Liu CM,Liu DP,et al.MicroRNAs:key participants in gene regulatory networks[J].Curr Opin Chem Biol,2003,7(4):516-523.
[4] Carrington JC,Ambros V.Role of microRNAs in plant and animal development[J].Science,2003,301(5631):336-348.
[5] McManus MT.MicroRNAs and cancer[J].Semin Cancer Biol,2003,13(4):253-268.
[6] Esau C,Kang X,Peralta E,et al.MicroRNA-143 regulates adipocyte differentiation[J].J Biol Chem,2004,279(50):52361-52375.
[7] McGregor RA,Choi MS.microRNAs in the regulation of adipogenesis and obesity[J].Curr Mol Med,2011,11(4):304-316.
[8] Lee RC,Feinbaum RL,Ambros V.The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843-854.
[9] miR Base:the microRNA database.http://www.mirbase.org/
[10] Pencheva N,Tran H,Buss C,et al.Convergent multi-miRNA targeting of ApoE drives LRP1/LRP8-dependent melanoma metastasis and angiogenesis[J].Cell,2012,151(5):1068-1082.
[11] Frecha C,Szecsi J,Cosset FL,et al.Strategies for targeting lentiviral vectors[J].Curr Gene Ther,2008,8(6):449-460.
[12] 史春梅,陈玲,徐广峰,等.人miR-17-92 cluster慢病毒载体构建及其在人脂肪细胞的表达验证[J].江苏医药,2012,38(6):624-627.
基金
国家自然科学基金(81100618)；江苏省自然科学基金(BK2011107)；南京市科技发展计划(201104013)
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