Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Cloning of short hairpin RNAs for gene knockdown in mammalian cells

Nature Methods volume 1, pages 163–167 (2004)Cite this article

  • Amplification buffer (NEB; provided with Vent DNA polymerase) and Vent DNA polymerase (NEB)

  • GC-melt PCR reagent (Clontech; provided with Advantage-GC PCR kit)

  • Dimethyl sulfoxide (DMSO; Sigma)

  • dNTP solution (10 mM)

  • Primer oligonucleotides (the sequences shown in Fig. 3) and Template oligonucleotide (as described in step 1; example in Fig. 2)

  • Phenol/chloroform, 1:1 (vol/vol), and 10% chloroform

  • 0.3 M sodium acetate (pH 4.8)

  • 70% (vol/vol) ethanol

  • Glycogen carrier (Boehringer, Mannheim)

  • High-melting-temperature agarose (such as MetaPhor Agarose, Cambrex)

    Glass bead kit (such as QiaexII gel extraction kit, Qiagen)

    Restriction endonucleases EcoRI and XhoI

    PirPlus (PIR1)-competent bacteria (Open Biosystems) and selective antibiotics (chloramphenicol and kanamycin)

    T4 DNA ligase (NEB, high-concentration ligase)

    Vector: pSM2 (pShag Magic Version 2.0; Open Biosystems)

Amplification buffer (NEB; provided with Vent DNA polymerase) and Vent DNA polymerase (NEB)

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Retroviral backbone of pSHAG-MAGIC2.
Figure 2: Design and sequence of a sample template.
Figure 3: Primer oligonucleotides for amplification.

References

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

    Article  CAS  Google Scholar 

  2. Paddison, P.J. & Hannon, G.J. siRNAs and shRNA: skeleton keys to the human genome. Curr. Opin. Mol. Ther. 5, 217–224 (2003).

    CAS  PubMed  Google Scholar 

  3. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  4. Caplen, N.J., Parrish, S., Imani, F., Fire, A. & Morgan, R.A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. 98, 9742–9747 (2001).

    Article  CAS  Google Scholar 

  5. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004).

    Article  CAS  Google Scholar 

  6. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    Article  CAS  Google Scholar 

  7. Paddison, P.J., Caudy, A.A. & Hannon, G.J. Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. 99, 1443–1448 (2002).

    Article  CAS  Google Scholar 

  8. Paddison, P., Caudy A.A., Bernstein, E., Hannon G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).

    Article  CAS  Google Scholar 

  9. McCaffrey, A.P. et al. RNA interference in adult mice. Nature 418, 38–39 (2002).

    Article  CAS  Google Scholar 

  10. Hemann, M.T. et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33, 396–400 (2003).

    Article  CAS  Google Scholar 

  11. Carmell, M.A., Zhang, L., Conklin, D.S., Hannon G.J. & Rosenquist, T.A. Germline transmission of RNAi in mice. Nat. Struct. Biol. 10, 91–92 (2003).

    Article  CAS  Google Scholar 

  12. Rubinson, D.A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406 (2003).

    Article  CAS  Google Scholar 

  13. Paddison, P.J., Caudy, A.A., Sachidanandam, R. & Hannon, G.J. Short hairpin activated gene silencing in mammalian cell. Methods Mol. Biol. 265, 85–100 (2004).

    CAS  PubMed  Google Scholar 

  14. Chen, C.Z., Li, L., Lodish, H.F. & Bartel, D.P. MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86 (2004).

    Article  CAS  Google Scholar 

  15. Brummelkamp, T.R., Bernards, R. & Agami, R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–247 (2002).

    Article  CAS  Google Scholar 

  16. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    Article  CAS  Google Scholar 

  17. Zeng, Y. & Cullen, B.R. Sequence requirements for micro RNA processing and function in human cells. RNA 9, 112–123 (2003).

    Article  CAS  Google Scholar 

  18. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  Google Scholar 

  19. Zeng, Y., Wagner, E.J. & Cullen, B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell. 9, 1327–1333 (2002).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA

    Patrick J Paddison, Jose Maria Silva, Kenneth Chang, Nihar Sheth, Ravi Sachidanandam & Gregory J Hannon

  2. Rosetta Inpharmatics, 401 Terry Avenue N., Seattle, 98109, Washington, USA

    Michele Cleary

Authors
  1. Patrick J Paddison
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michele Cleary
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jose Maria Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kenneth Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nihar Sheth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ravi Sachidanandam
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gregory J Hannon
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paddison, P., Cleary, M., Silva, J. et al. Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat Methods 1, 163–167 (2004). https://doi.org/10.1038/nmeth1104-163

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1104-163

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing