Abstract
MicroRNA-based short hairpin RNAs (shRNAs) are natural inducers of RNA interference and have been increasingly used in shRNA expression strategies. In the present study, we compared the efficiencies of exogenous green fluorescence protein (GFP) and endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown and red fluorescent protein (RFP) indicator expression mediated by three differently designed plasmids. RFP was introduced either at the 5′ end, at the 3′ end of the human mir155-based target gene (TG) (e.g., GFP or GAPDH) shRNA expression cassette (EC), or at the 3′ end of the chimeric intron-containing TG shRNA EC. Comparisons with the control vector showed an obvious reduction of GFP or GAPDH expression with the various shRNA expression plasmids (P < 0.05). When RFP was located at the 5′ end or at the 3′ end of the TG shRNA EC, RFP expression was low; whereas when RFP was connected with the chimeric intron-containing TG shRNA EC, RFP expression was high. Taken together, this study demonstrated an efficient plasmid design for both TG silencing induced by microRNA-based shRNA and indicator gene expression in vitro.
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References
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349
Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297:2056–2060
Cullen BR (2004) Transcription and processing of human microRNA precursors. Mol Cell 16:861–865
Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385
Cullen BR (2005) RNAi the natural way. Nat Genet 37:1163–1165
Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR, Sheth N, Bradshaw J, Burchard J, Kulkarni A, Cavet G, Sachidanandam R, McCombie WR, Cleary MA, Elledge SJ, Hannon GJ (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37:1281–1288
Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 102:13212–13217
Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37:1289–1295
Chung KH, Hart CC, Al-Bassam S, Avery A, Taylor J, Patel PD, Vojtek AB, Turner DL (2006) Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 34:e53
Shan Z, Lin Q, Deng C, Li X, Huang W, Tan H, Fu Y, Yang M, Yu XY (2008) An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs. Mol Biol Rep. doi: 10.1007/s11033-008-9339-8
Shan ZX, Lin QX, Yang M, Deng CY, Kuang SJ, Zhou ZL, Xiao DZ, Liu XY, Lin SG, Yu XY (2009) A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs. Mol Cell Biochem 323:81–89
Du G, Yonekubo J, Zeng Y, Osisami M, Frohman MA (2006) Design of expression vectors for RNA interference based on miRNAs and RNA splicing. FEBS J 273:5421–5427
Qiu L, Wang H, Xia X, Zhou H, Xu Z (2008) A construct with fluorescent indicators for conditional expression of miRNA. BMC Biotechnol 8:77
Hammond SM, Caudy AA, Hannon GJ (2001) Posttranscriptional gene silencing by double-stranded RNA. Nat Rev Genet 2:110–119
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Tsuchiya S, Okuno Y, Tsujimoto G (2006) MicroRNA: biogenetic and functional mechanisms and involvements in cell differentiation and cancer. J Pharmacol Sci 101:267–270
Buchman AR, Berg P (1988) Comparison of intron-dependent and intron independent gene expression. Mol Cell Biol 8:4395–4405
Huang MTF, Gorman CM (1990) Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucl Acids Res 18:937–947
Liu YP, Haasnoot J, ter Brake O, Berkhout B, Konstantinova P (2008) Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res 36:2811–2824
Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (No. 30672077, 30772142), the Natural Science Foundation of the Guangdong Province (No. 06020831) and the National Key Basic Research Program (NKBRP) of China (No. 2006CB503806).
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Shan, Z.X., Lin, Q.X., Deng, C.Y. et al. Comparison of approaches for efficient gene silencing induced by microRNA-based short hairpin RNA and indicator gene expression. Mol Biol Rep 37, 1831–1839 (2010). https://doi.org/10.1007/s11033-009-9618-z
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DOI: https://doi.org/10.1007/s11033-009-9618-z