Skip to main content

Advertisement

SpringerLink
Log in

Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 ± 5.06% for CMVE-U6 promoter group and 88.26 ± 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 ± 3.06% and 51.56 ± 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 ± 2.96% for CMVE-U6 promoter group and 83.06 ± 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

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 

  • Arendt CW, Tang G, Zilberstein A (2003) Vector systems for the delivery of small interfering RNAs: managing the RISC. Chembiochemistry 4:1129–1136

    Article  CAS  Google Scholar 

  • Bark C, Weller P, Zabielski J, Janson L, Pettersson U (1987) A distant enhancer element is required for polymerase III transcription of a U6 RNA gene. Nature 328:356–359

    Article  PubMed  CAS  Google Scholar 

  • Boden D, Pusch O, Lee F, Tucker L, Shank PR, Ramratnam B (2003) Promoter choice affects the potency of HIV-1 specific RNA interference. Nucleic Acids Res 31:5033–5038

    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 

  • Carbon P, Murgo S, Ebel JP, Krol A, Tebb G, Mattaj LW (1987) A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II. Cell 51:71–79

    Article  PubMed  CAS  Google Scholar 

  • Das G, Henning D, Wright D, Reddy R (1988) Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. EMBO J 7:503–512

    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 

  • Dorn G, Patel S, Wotherspoon G, Hemmings-Mieszczak M, Barclay J, Natt FJ, Martin P, Bevan S, Fox A (2004) siRNA relieves chronic neuropathic pain. Nucleic Acids Res 32:e49

    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 

  • 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 

  • Hassani Z, Francois J, Alfama G, Dubois GM, Paris M, Giovannangeli C, Demeneix BA (2007) A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain.Nucleic Acids Res 35:e65

    Article  CAS  Google Scholar 

  • Hernandez N (2001) Small nuclear RNA genes: a model system to study fundamental mechanisms of transcription. J Biol Chem 276:26733–26736

    Article  PubMed  CAS  Google Scholar 

  • Hutvagner G, Zamore PD (2002) RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12:225–232

    Article  PubMed  CAS  Google Scholar 

  • Ilves H, Barske C, Junker U, Bohnlein E, Veres G (1996) Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study. Gene 171:203–208

    Article  PubMed  CAS  Google Scholar 

  • Jacque JM, Triques K, Stevenson M (2002) Modulation of HIV-1 replication by RNA interference. Nature 418:435–438

    Article  PubMed  CAS  Google Scholar 

  • Koper-Emde D, Herrmann L, Sandrock B, BeNecke BJ (2004) RNA interference by small hairpin RNAs synthesized under control of the human 7S K RNA promoter. Biol Chem 385:791–794

    Article  PubMed  CAS  Google Scholar 

  • Kunkel GR, Pederson T (1988) Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Genes Dev 2:196–204

    Article  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, 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 

  • 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 9:420–428

    Article  PubMed  CAS  Google Scholar 

  • Lobo SM, Hernandez N (1989) A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell 58:55–67

    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 

  • Mattaj IW, Dathan NA, Parry HD, Carbon P, Krol A (1988) Changing the RNA polymerase specificity of U snRNA gene promoters. Cell 55:435–442

    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 

  • Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508

    Article  PubMed  CAS  Google Scholar 

  • Song J, Pang S, Lua Y, Chiu R (2004) Poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNA from a pol II promoter. Biochem Biophys Res Commun 323:573–578

    Article  PubMed  CAS  Google Scholar 

  • Su J, Zhu Z, Wang Y, Xiong F, Zou J (2007) The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo. Mar Biotechnol DOI 10.1007/s10126-007-9059-4

  • Wang N (2007) Study of short interfering RNA (siRNA) produced in transcription system of zebrafish embryo. Dissertation of doctor of science, Chinese Academy of Sciences

  • 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 

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

    Article  PubMed  CAS  Google Scholar 

  • Xia XG, Zhou H, Ding H, Affar EB, Shi Y, Xu Z (2003) An enhanced U6 promoter for synthesis of short hairpin RNA. Nucleic Acids Res 31:e100

    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 

Download references

Acknowledgements

The authors thank Ming Li for microinjection. We also appreciate Wei Hu, Jun Dai, Na Wang, Shangping Chen, and other laboratory members for technical assistance and helpful discussion. This work was supported by grants from National Natural Science Foundation of China (30740009, 30540084 and 30428024), from 973 National Basic Research Program of China (2006CB102100), from Chinese Academy of Sciences (KSCX2-YW-N-021), from Northwest A & F University in China (08080262, 08080245 and 01140508), from China Postdoctoral Science Foundation (20070410298) and from Institute of Hydrobiology, CAS (2007FB09).

Author information

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, Feng Xiong & Yaping Wang

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

    Jianguo Su

Authors
  1. Jianguo Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zuoyan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Feng Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yaping Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zuoyan Zhu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Su, J., Zhu, Z., Xiong, F. et al. Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish. Mar Biotechnol 10, 511–517 (2008). https://doi.org/10.1007/s10126-008-9087-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-008-9087-8

Keywords

Search

Navigation