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Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs

Gene Therapy volume 15, pages 191–202 (2008)Cite this article

Abstract

To induce RNA interference (RNAi), either small interfering RNAs (siRNAs) are directly introduced into the cell or short hairpin RNAs (shRNAs) are expressed from a DNA vector. At present, shRNAs are commonly synthesized by RNA polymerase III (Pol III) promoters of the H1 and U6 RNAs. In this study, we designed and characterized a new set of plasmid vectors driven by promoters of the Epstein–Barr virus (EBV)-encoded small RNAs (EBERs). The EBERs are the most abundant transcript in infected cells and they are transcribed by Pol III. We showed that the EBER promoters were able to drive the expression of shRNA fusion transcripts. siRNAs processed from these fusion transcripts specifically and effectively inhibited the expression of homologous reporter or endogenous genes in various types of cells. The partial EBER sequences in the fusion transcripts did not activate double-stranded RNA-dependent protein kinase or suppress RNAi. In nasopharyngeal carcinoma cells, the EBER2 promoter was stronger than the H1 and U6 promoters in shRNA synthesis, leading to more effective knockdown of the target genes. Taken together, our findings suggest that the EBER promoters fundamentally different from those of H1 and U6 can be used to drive the intracellular expression of shRNAs for effective silencing of target genes in mammalian cells and particularly, in EBV-infected cells.

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Acknowledgements

We thank GJ Hannon, R Agami and IW Mattaj for reagents; KY Yuen for support with quantitative PCR analysis; C-P Chan, K-T Chin and K-L Siu for technical advice; and Y-P Ching, K-T Chin, ACS Chun and Y-T Siu for critical reading of the manuscript. This work was supported by Innovation and Technology Fund of Hong Kong (ITS/112/02), Fogarty International Center of National Institutes of Health (R01 TW06186-01), Hong Kong Research Grants Council (HKU 1/06C), and University of Hong Kong (seed funding program for basic research 2004).

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  1. Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, Hong Kong, China

    E Y-W Choy, K-H Kok & D-Y Jin

  2. Department of Anatomy, Faculty of Medicine, The University of Hong Kong, Hong Kong, China

    S W Tsao

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Correspondence to D-Y Jin.

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Choy, EW., Kok, KH., Tsao, S. et al. Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs. Gene Ther 15, 191–202 (2008). https://doi.org/10.1038/sj.gt.3303055

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