Protocol | Published:

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

Nature Protocols volume 4, pages 13381348 (2009) | Download Citation

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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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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.

Author information

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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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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DOI

https://doi.org/10.1038/nprot.2009.145

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