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High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion

Nature Methods volume 2, pages 967–973 (2005)Cite this article

Abstract

Use of RNA interference (RNAi) as a reverse genetics tool for silencing genes in mammalian cells is achieved by in vitro transfection of small interfering RNAs (siRNAs). For a target gene, several siRNAs must be designed according to the empirical rules. We demonstrated that functional short hairpin RNAs (shRNAs) could be synthesized in Escherichia coli and delivered directly via bacterial invasion to the near entirety of a mammalian cell population to trigger RNAi. Furthermore, using a luciferase–target gene transcript, we identified effective shRNAs and siRNAs from RNAi libraries delivered conveniently through bacterial invasion in 96-well plates without need for preparation, purification and transfection of shRNAs. Notably, several of the most highly effective shRNAs and siRNAs identified do not fit the empirical rules commonly used for siRNA design, suggesting that this approach is a powerful tool for RNAi research, and could be used complementarily to the empirical rules for RNAi applications.

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Figure 1: Expression of shRNAs in E. coli.
Figure 2: Schematic of the protocol for screening of effective shRNAs in 96-well plates.
Figure 3: Functional delivery of shRNAs via invasive bacteria to mammalian cells.
Figure 4: High-throughput screens for effective shRNAs.
Figure 5: Evaluation of RNAi potency by dose response assay in silencing the endogenous MVP gene.

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Acknowledgements

We thank C. Grillot-Courvalin for providing the invasin gene–containing plasmid pGBΩinv-hly and N. Fotouhi for providing an siRNA sequence. We also thank Y. Fortin and L. Pan for their excellent technical assistance, and M. Slater and K. Bijian for their valuable comments on the manuscript.

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Authors and Affiliations

  1. Health Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, H4P 2R2, Québec, Canada

    Hui-Fen Zhao, Denis L'Abbé, Normand Jolicoeur, Meiqun Wu, Zhen Li, Zhenbao Yu & Shi-Hsiang Shen

  2. Department of Medicine, McGill University, Montréal, H3G 1A4, Québec, Canada

    Shi-Hsiang Shen

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  1. Hui-Fen Zhao
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Correspondence to Hui-Fen Zhao or Shi-Hsiang Shen.

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Supplementary information

Supplementary Fig. 1

Targeting of shRNAs to the MVP region of the hybrid Luc-MVP transcript led to the destruction of the entire transcript. (PDF 148 kb)

Supplementary Fig. 2

Bacterial dose responses (MOI) for evaluation of effective shRNA concentrations in invasion. (PDF 143 kb)

Supplementary Fig. 3

High throughput screens for effective shRNAs from shSHP and shPTEN libraries. (PDF 189 kb)

Supplementary Table 1

siRNA and shRNA sequences used in the experiments. (PDF 69 kb)

Supplementary Table 2

Efficiency of gene transfection in conjunction with the age of bacterial culture. (PDF 61 kb)

Supplementary Table 3

shRNA sequences identified in screening from the RNAi libraries. (PDF 62 kb)

Supplementary Methods (DOC 25 kb)

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Zhao, HF., L'Abbé, D., Jolicoeur, N. et al. High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion. Nat Methods 2, 967–973 (2005). https://doi.org/10.1038/nmeth812

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