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The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo

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Abstract

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 ± 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 ± 4% and decreased the level of their corresponding mRNAs up to 54.52 ± 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

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References

  • Acosta J, Carpio Y, Borroto I, Gonzalez O, Estrada MP (2005) Myostatin gene silenced by RNAi show a zebrafish giant phenotype. J Biotechnol 119:324-331

    Article  PubMed  CAS  Google Scholar 

  • Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268-272

    Article  PubMed  CAS  Google Scholar 

  • Banan M, Puri N (2004) The ins and outs of RNAi in mammalian cells. Curr Pharm Biotechnol 5:441-450

    Article  PubMed  CAS  Google Scholar 

  • Boonanuntanasarn S, Yoshizaki G, Takeuchi T (2003) Specific gene silencing using small interfering RNAs in fish embryos. Biochem Biophys Res Commun 310:1089-1095

    Article  PubMed  CAS  Google Scholar 

  • Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550-553

    Article  PubMed  CAS  Google Scholar 

  • Ding S, Li H, Lu R, Li F, Li W (2004) RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res 102:109-115

    Article  PubMed  CAS  Google Scholar 

  • Dodd A, Chambers SP, Love DR (2004) Short interfering RNA-mediated gene targeting in the zebrafish. FEBS Lett 561:89-93

    Article  PubMed  CAS  Google Scholar 

  • Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457-467

    Article  PubMed  CAS  Google Scholar 

  • Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Geiduschek EP, Kassavetis GA (2001) The RNA polymerase III Transcription Apparatus. J Mol Biol 310:1-26

    Article  PubMed  CAS  Google Scholar 

  • Gil J, Esteban M (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5:107-114

    Article  PubMed  CAS  Google Scholar 

  • Halpern ME, Ho RK, Walker C, Kimmel CB (1993) Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 75:99-111

    PubMed  CAS  Google Scholar 

  • Hannon GJ (2002) RNA interference. Nature 418:244-251

    Article  PubMed  CAS  Google Scholar 

  • Jonathan EP, Foster JS, Kestler D, Alan S, Wall JS (2007) Inhibition of Bence-Jones protein synthesis by RNA interference. J Immunol 178:S88-S89

    Google Scholar 

  • Kondraganti S, Gondi CS, McCutcheon I, Dinh DH, Gujrati M, Rao JS, Olivero WC (2006) RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth. Int J Oncol 28:1353-1360

    PubMed  CAS  Google Scholar 

  • Liu J, Sun Y, Wang N, Wang Y, Zhu Z (2006) Upstream regulatory region of zebrafish lunatic fringe: isolation and promoter analysis. Mar Biotechnol 8:357-365

    Article  PubMed  CAS  Google Scholar 

  • Li YX, Farrell MJ, Liu R, Mohanty N, Kirby ML (2000) Double-stranded RNA injection produces null phenotypes in zebrafish. Dev Biol 217:394-405

    Article  PubMed  CAS  Google Scholar 

  • Liu WY, Wang Y, Qin Y, Wang YP, Zhu ZY (2007) Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant lox sites. Mar Biotechnol 9:418-420

    CAS  Google Scholar 

  • Liu WY, Wang Y, Sun YH, Wang Y, Wang YP, Chen SP, Zhu ZY (2005) Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase. Dev Growth Differ 47:323-331

    Article  PubMed  CAS  Google Scholar 

  • Mangos S, Vanderbeld B, Krawetz R, Sudol K, Kelly GM (2001) Ran binding protein RanBP1 in zebrafish embryonic development. Mol Reprod Dev 59:235-248

    Article  PubMed  CAS  Google Scholar 

  • McIntyre GJ, Fanning GC (2006) Design and cloning strategies for constructing shRNA expression vectors. BMC Biotechnol 6:1-8

    Article  PubMed  CAS  Google Scholar 

  • Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2:279-289

    Article  PubMed  CAS  Google Scholar 

  • Oates AC, Bruce AE, Ho RK (2000) Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Dev Biol 224:20-28

    Article  PubMed  CAS  Google Scholar 

  • Sarmasik A, Warr G, Chen T (2002) Production of transgenic medaka with increased resistance to bacterial pathogens. Mar Biotechnol 4:310-322

    Article  PubMed  CAS  Google Scholar 

  • Schramm L, Hernandez N (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev 16:2593-2620

    Article  PubMed  CAS  Google Scholar 

  • Shinagawa T, Ishii S (2003) Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes Dev 17:1340-1345

    Article  PubMed  CAS  Google Scholar 

  • Tiscornia G, Singer O, Ikawa M, Verma IM (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 100:1844-1848

    Article  PubMed  CAS  Google Scholar 

  • Tuschl T (2002) Expanding small RNA interference. Nat Biotechnol 20:446-448

    Article  PubMed  CAS  Google Scholar 

  • Wang N, Sun Y, Liu J, Wu G, Su J, Wang Y, Zhu Z (2007) Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system. J Biomed Sci 14:767-776

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Wu G, Yu L, Yuan J, Fang F, Zhai Z, Wang F, Wang H (2006) Inhibition of CD147 expression reduces tumor cell invasion in human prostate cancer cell line via RNA interference. Cancer Biol Ther 5:608-614

    Article  PubMed  CAS  Google Scholar 

  • Wargelius A, Ellingsen S, Fjose A (1999) Double-stranded RNA induces specific developmental defects in zebrafish embryos. Biochem Biophys Res Commun 263:156-161

    Article  PubMed  CAS  Google Scholar 

  • Wu Y, Zhang G, Xiong Q, Luo F, Cui C, Hu W, Yu Y, Su J, Xu A, Zhu Z (2006) Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin. Mar Biotechnol 8:304-311

    Article  PubMed  CAS  Google Scholar 

  • Xia H, Mao Q, Paulson H, Davidson B (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20:1006-1010

    Article  PubMed  CAS  Google Scholar 

  • Xie J, Lu L, Deng M, Weng S, Zhu J, Wu Y, Gan L, Chan SM, He J (2005) Inhibition of reporter gene and Iridovirus-tiger frog virus in fish cell by RNA interference. Virology 338:43-52

    Article  PubMed  CAS  Google Scholar 

  • Yoder JA, Nielsen ME, Amemiya CT, Litman GW (2002) Zebrafish as an immunological model system. Microbes Infect 4:1469-1478

    Article  PubMed  CAS  Google Scholar 

  • Yuan J, Wang X, Zhang Y, Hu X, Deng X, Fei J, Li N (2006) shRNA transcribed by RNA Pol II promoter induce RNA interference in mammalian cell. Mol Biol Rep 33:43-49

    Article  PubMed  CAS  Google Scholar 

  • Zhao Z, Cao Y, Li M, Meng A (2001) Double-stranded RNA injection produces nonspecific defects in zebrafish. Dev Biol 229:215-223

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors thank Miss Yuejiao Lu for critically reading the manuscript. The technical assistance provided by Ming Li, Na Wang, Jun Dai, Shangping Chen, and other laboratory members was greatly appreciated. This work was supported by (30428024 and 30540084) from National Natural Science Foundation of China, (KSCX2-YW-N-021) from Chinese Academy of Sciences, (08080262, 08080245, and 01140508) from Northwest A & F University in China, (20070410298) from China Postdoctoral Science Foundation and (2007FB09) from Institute of Hydrobiology, CAS.

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Authors and Affiliations

  1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China

    Jianguo Su, Zuoyan Zhu, Yaping Wang & Feng Xiong

  2. Department of Aquaculture, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China

    Jianguo Su

  3. School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, United Kingdom

    Jun Zou

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  1. Jianguo Su
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  2. Zuoyan Zhu
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  3. Yaping Wang
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  4. Feng Xiong
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  5. Jun Zou
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Correspondence to Zuoyan Zhu.

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Su, J., Zhu, Z., Wang, Y. et al. The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo. Mar Biotechnol 10, 262–269 (2008). https://doi.org/10.1007/s10126-007-9059-4

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  • DOI: https://doi.org/10.1007/s10126-007-9059-4

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