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Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression

Nature Protocols volume 4, pages 1338–1348 (2009)Cite this article

Abstract

Small hairpin RNA (shRNA) is a powerful tool for inhibiting gene expression. One limitation has been that this technique has been used primarily to target a single gene. This protocol expands upon previous methods by describing a knockdown vector that facilitates cloning of multiple shRNAs; this allows targeted knockdown of more than one gene or of a single gene that may otherwise be difficult to knockdown using a single shRNA. The targeted gene(s) can be readily re-expressed by transfecting knockdown cells with a knock-in vector, containing an shRNA-refractive cDNA that will express the protein-of-interest even in the presence of shRNAs. The constructed knockdown and knock-in vectors can be easily used concurrently to assess possible interrelationships between genes, the effects of gene loss on cell function and/or their restoration by replacing targeted genes one at a time. The entire knockdown or knock-in procedure can be completed in ∼3–4 months.

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Figure 1
Figure 2: Schematic representation of cloning tandem, multi-shRNA sequences.
Figure 3: Design of shRNA oligonucleotides for knocking down gene expression and silent mutations for knocking in gene expression at internal gene regions.
Figure 4: Analysis of targeted mRNAs in transfected cells encoding knockdown vectors; northern blotting of PSTK and SECp43 in shRNA transfected NIH 3T3 cells.
Figure 5: Autoradiogram of 75Se-labeled selenoproteins in double knockdown or knock-in cells.

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References

  1. Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Novina, C.D. & Sharp, P.A. The RNAi revolution. Nature 430, 161–164 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Xu, X.M. et al. Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery. J. Biol. Chem. 280, 41568–41575 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Jazag, A. et al. Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components. Nucleic Acids Res. 33, e131 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  5. ter, B.O., Konstantinova, P., Ceylan, M. & Berkhout, B. Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol. Ther. 14, 883–892 (2006).

    Article  Google Scholar 

  6. Wang, S., Shi, Z., Liu, W., Jules, J. & Feng, X. Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing. BMC. Biotechnol. 6, 50 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Gou, D. et al. A novel approach for the construction of multiple shRNA expression vectors. J. Gene Med. 9, 751–763 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Dafny-Yelin, M., Chung, S.M., Frankman, E.L. & Tzfira, T. pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants. Plant Physiol 145, 1272–1281 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Song, J., Giang, A., Lu, Y., Pang, S. & Chiu, R. Multiple shRNA expressing vector enhances efficiency of gene silencing. BMB. Rep. 41, 358–362 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Yan, Y., Zhang, J., Guo, J.L., Huang, W. & Yang, Y.Z. Multiple shRNA-mediated knockdown of TACE reduces the malignancy of HeLa cells. Cell Biol. Int. 33, 158–164 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Xu, X.M. et al. Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis. Biochem. J. 404, 115–120 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jiang, Y. & Price, D.H. Rescue of the TTF2 knockdown phenotype with an siRNA-resistant replacement vector. Cell Cycle 3, 1151–1153 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Kim, D.H. & Rossi, J.J. Coupling of RNAi-mediated target downregulation with gene replacement. Antisense Nucleic Acid Drug Dev. 13, 151–155 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. O'Reilly, M. et al. RNA interference-mediated suppression and replacement of human rhodopsin in vivo . Am. J. Hum. Genet. 81, 127–135 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jallow, Z., Jacobi, U.G., Weeks, D.L., Dawid, I.B. & Veenstra, G.J. Specialized and redundant roles of TBP and a vertebrate-specific TBP paralog in embryonic gene regulation in Xenopus. Proc. Natl. Acad. Sci. USA 101, 13525–13530 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Unwalla, H.J. et al. Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein. J. Virol. 80, 1863–1873 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Laatsch, A., Ragozin, S., Grewal, T., Beisiegel, U. & Joerg, H. Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP). Eur. J. Cell Biol. 83, 113–120 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Yoo, M.H. et al. A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3′-UTR. RNA 13, 921–929 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Low, S.C. & Berry, M.J. Knowing when not to stop: selenocysteine incorporation in eukaryotes. Trends Biochem. Sci. 21, 203–208 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Kimchi-Sarfaty, C. et al. A 'silent' polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525–528 (2007).

    Article  CAS  Google Scholar 

  21. Hsieh, A.C. et al. A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. Nucleic Acids Res. 32, 893–901 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Krueger, U. et al. Insights into effective RNAi gained from large-scale siRNA validation screening. Oligonucleotides 17, 237–250 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Kiang, A. et al. Fully deleted adenovirus persistently expressing GAA accomplishes long-term skeletal muscle glycogen correction in tolerant and nontolerant GSD-II mice. Mol. Ther. 13, 127–134 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Sano, M., Kato, Y., Akashi, H., Miyagishi, M. & Taira, K. Novel methods for expressing RNA interference in human cells. Methods Enzymol. 392, 97–112 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Scherer, L.J., Frank, R. & Rossi, J.J. Optimization and characterization of tRNA-shRNA expression constructs. Nucleic Acids Res. 35, 2620–2628 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. An, D.S. et al. Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol. Ther. 14, 494–504 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Makinen, P.I. et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain. J. Gene Med. 8, 433–441 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Bannister, S.C., Wise, T.G., Cahill, D.M. & Doran, T.J. Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression. BMC. Biotechnol. 7, 79 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lambeth, L.S., Wise, T.G., Moore, R.J., Muralitharan, M.S. & Doran, T.J. Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression. Anim. Genet. 37, 369–372 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Koper-Emde, D., Herrmann, L., Sandrock, B. & Benecke, B.J. RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter. Biol. Chem. 385, 791–794 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Kawasaki, H. & Taira, K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res. 31, 700–707 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Paul, C.P. et al. Localized expression of small RNA inhibitors in human cells. Mol. Ther. 7, 237–247 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537–541 (2006).

    Article  CAS  Google Scholar 

  34. ter, B.O. et al. Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol. Ther. 16, 557–564 (2008).

    Article  Google Scholar 

  35. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Yoo, M.H., Xu, X.M., Carlson, B.A., Gladyshev, V.N. & Hatfield, D.L. Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells. J. Biol. Chem. 281, 13005–13008 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Birmingham, A. et al. 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Svoboda, P. Off-targeting and other non-specific effects of RNAi experiments in mammalian cells. Curr. Opin. Mol. Ther. 9, 248–257 (2007).

    CAS  PubMed  Google Scholar 

  41. Novoselov, S.V. et al. Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern. J. Biol. Chem. 282, 11960–11968 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Yoo, M.H. et al. Targeting thioredoxin reductase 1 reduction in cancer cells inhibits self-sufficient growth and DNA replication. PLoS. ONE. 2, e1112 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Sambrook, J. & Russell, D.W. Extraction, purification and analysis of mRNA from eukaryotic cells. in Molecular Cloning–A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) 1–94 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

    Google Scholar 

  44. Ding, F. & Grabowski, P.J. Identification of a protein component of a mammalian tRNA(Sec) complex implicated in the decoding of UGA as selenocysteine. RNA 5, 1561–1569 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Carlson, B.A. et al. Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase. Proc. Natl. Acad. Sci. USA 101, 12848–12853 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Xu, X.M. et al. Biosynthesis of selenocysteine on its tRNA in eukaryotes. PLoS. Biol. 5, 96–105 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), NCI, Center for Cancer Research to D.L.H. and NIH grants to V.N.G.

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Authors and Affiliations

  1. Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Xue-Ming Xu, Min-Hyuk Yoo, Bradley A Carlson & Dolph L Hatfield

  2. Department of Biochemistry, University of Nebraska and Redox Biology Center, Lincoln, Nebraska, USA

    Vadim N Gladyshev

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  1. Xue-Ming Xu
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Contributions

X.-M.X., designed and constructed the pU6-m4 vector; X.-M.X. and M.-H.Y. designed and constructed shRNA and knock-in constructs; X.-M.X., M.-H.Y. and B.A.C. designed and carried out the experiments with the advice of D.L.H. X.-M.X., M.-H.Y., B.A.C., V.N.G. and D.L.H. analyzed results, interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Dolph L Hatfield.

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Xu, XM., Yoo, MH., Carlson, B. et al. Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression. Nat Protoc 4, 1338–1348 (2009). https://doi.org/10.1038/nprot.2009.145

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