Skip to main content
SpringerLink
Log in

shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

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

Abbreviations

shRNA:

Short hairpin RNA

RNAi:

RNA interference

IR:

Inverted repeat

Nt:

Nucleotides

siRNA:

Short interference RNA

dsRNA:

Double-stranded RNA

PCR:

Polymerase chain reaction

Rluc:

Renilla luciferase

EGFP:

Enhanced green fluorescent protein

References

  1. Agrawal, N., Dasaradhi, P. V., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA interference: Biology, mechanism, and applications. Microbiology and Molecular Biology Reviews, 67, 657–685. doi:10.1128/MMBR.67.4.657-685.2003.

    Article  CAS  Google Scholar 

  2. Dawe, R. K. (2003). RNA interference, transposons, and the centromere. The Plant Cell, 15, 297–301. doi:10.1105/tpc.150230.

    Article  CAS  Google Scholar 

  3. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811. doi:10.1038/35888.

    Article  CAS  Google Scholar 

  4. Hannon, G. J. (2002). RNA interference. Nature, 418, 244–251. doi:10.1038/418244a.

    Article  CAS  Google Scholar 

  5. Denli, A. M., & Hannon, G. J. (2003). RNAi: An ever-growing puzzle. Trends in Biochemical Sciences, 28, 196–201. doi:10.1016/S0968-0004(03)00058-6.

    Article  CAS  Google Scholar 

  6. McManus, M. T., & Sharp, P. A. (2002). Gene silencing in mammals by small interfering RNAs. Nature Reviews. Genetics, 3, 737–747. doi:10.1038/nrg908.

    Article  CAS  Google Scholar 

  7. Zamore, P. D. (2002). Ancient pathways programmed by small RNAs. Science, 296, 1265–1269. doi:10.1126/science.1072457.

    Article  CAS  Google Scholar 

  8. Hammond, S. M., Bernstein, E., Beach, D., & Hannon, G. J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293–296. doi:10.1038/35005107.

    Article  CAS  Google Scholar 

  9. Brummelkamp, T. R., Bernards, R., & Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553. doi:10.1126/science.1068999.

    Article  CAS  Google Scholar 

  10. Dykxhoorn, D. M., Novina, C. D., & Sharp, P. A. (2003). Killing the messenger: Short RNAs that silence gene expression. Nature Reviews. Molecular Cell Biology, 4, 457–467. doi:10.1038/nrm1129.

    Article  CAS  Google Scholar 

  11. Konet, D. S., Anderson, J., Piper, J., Akkina, R., Suchman, E., & Carlson, J. (2007). Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells. Insect Molecular Biology, 16, 199–206. doi:10.1111/j.1365-2583.2006.00714.x.

    Article  CAS  Google Scholar 

  12. Wakiyama, M., Matsumoto, T., & Yokoyama, S. (2005). Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells. Biochemical and Biophysical Research Communications, 331, 1163–1170. doi:10.1016/j.bbrc.2005.03.240.

    Article  CAS  Google Scholar 

  13. Isobe, R., Sahara, K., Asano, S., & Bando, H. (2002). Antisense and double-strand RNA interference in a silkworm ovarian cell line. Journal of Insect Biotechnology and Sericology, 71, 43–47.

    CAS  Google Scholar 

  14. Katsuma, S., Mita, K., & Shimada, T. (2007). ERK- and JNK-dependent signaling pathways contribute to Bombyx mori nucleopolyhedrovirus infection. Journal of Virology, 81, 13700–13709. doi:10.1128/JVI.01683-07.

    Article  CAS  Google Scholar 

  15. Tsukioka, H., Takahashi, M., Mon, H., Okano, K., Mita, K., Shimada, T., et al. (2006). Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA. Nucleic Acids Research, 34, 1092–1101. doi:10.1093/nar/gkj507.

    Article  CAS  Google Scholar 

  16. Hernandez, G., Jr, Valafar, F., & Stumph, W. E. (2007). Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity. Nucleic Acids Research, 35, 21–34. doi:10.1093/nar/gkm298.

    Article  CAS  Google Scholar 

  17. Miyagishi, M., Sumimoto, H., Miyoshi, H., Kawakami, Y., & Taira, K. (2004). Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. The Journal of Gene Medicine, 6, 715–723. doi:10.1002/jgm.556.

    Article  CAS  Google Scholar 

  18. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., et al. (2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Research, 32, 936–948. doi:10.1093/nar/gkh247.

    Article  CAS  Google Scholar 

  19. Li, L., Lin, X., Khvorova, A., Fesik, S. W., & Shen, Y. (2007). Defining the optimal parameters for hairpin-based knockdown constructs. RNA (New York, NY), 13, 1765–1774. doi:10.1261/rna.599107.

    CAS  Google Scholar 

  20. Kanginakudru, S., Royer, C., Edupalli, S. V., Jalabert, A., Mauchamp, B., Prasad, S. V., et al. (2007). Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms. Insect Molecular Biology, 16, 635–644.

    CAS  Google Scholar 

  21. Quan, G. X., Kanda, T., & Tamura, T. (2002). Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene. Insect Molecular Biology, 11, 217–222. doi:10.1046/j.1365-2583.2002.00328.x.

    Article  CAS  Google Scholar 

  22. Tanaka, H., Yamamoto, M., Moriyama, Y., Yamao, M., Furukawa, S., Sagisaka, A., et al. (2005). A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori. Biochimica et Biophysica Acta, 1730, 10–21.

    CAS  Google Scholar 

  23. Tanaka, H., Matsuki, H., Furukawa, S., Sagisaka, A., Kotani, E., Mori, H., et al. (2007). Identification and functional analysis of Relish homologs in the silkworm, Bombyx mori. Biochimica et Biophysica Acta, 1769, 559–568.

    CAS  Google Scholar 

  24. Sano, M., Kato, Y., Akashi, H., Miyagishi, M., & Taira, K. (2005). Novel methods for expressing RNA interference in human cells. Methods in Enzymology, 392, 97–112. doi:10.1016/S0076-6879(04)92006-X.

    Article  CAS  Google Scholar 

  25. Castanotto, D., & Scherer, L. (2005). Targeting cellular genes with PCR cassettes expressing short interfering RNAs. Methods in Enzymology, 392, 173–185. doi:10.1016/S0076-6879(04)92010-1.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Genebank of the National Institute of Agrobiological Sciences for providing cell line. This work was supported by a grant from PRO-BRAIN.

Author information

Authors and Affiliations

  1. Innate Immunity Research Unit, National Institute of Agrobiological Sciences, Ibaraki, 305-8634, Japan

    Hiromitsu Tanaka, Kosuke Fujita, Aki Sagisaka, Kazuya Tomimoto & Minoru Yamakawa

  2. Genebank, National Institute of Agrobiological Sciences, Ibaraki, 305-8634, Japan

    Shigeo Imanishi

  3. Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305–8572, Japan

    Minoru Yamakawa

Authors
  1. Hiromitsu Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kosuke Fujita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aki Sagisaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuya Tomimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shigeo Imanishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Minoru Yamakawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiromitsu Tanaka.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tanaka, H., Fujita, K., Sagisaka, A. et al. shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line. Mol Biotechnol 41, 173–179 (2009). https://doi.org/10.1007/s12033-008-9108-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-008-9108-x

Keywords

Search

Navigation