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A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation

Gene Therapy volume 19, pages 1058–1064 (2012)Cite this article

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Abstract

RNA interference (RNAi) is a sequence-specific gene silencing mechanism with therapeutic potential against many human pathogens. To obtain a durable therapeutic effect, stable transduction of target cells with for instance a lentiviral vector that expresses a short hairpin (shRNA) inducer of the RNAi pathway is necessary. Apart from the intended therapeutic effect, this treatment can induce negative effects on cell proliferation via off-target effects. A careful evaluation of the transduced cells is required to develop a safe gene therapy approach. Stably transduced cells are usually selected by expression of the enhanced green fluorescent protein (GFP) marker. In this study we show that the mixed transduction culture, containing both transduced GFP+ and untransduced GFP− cells, can simply be passaged to score the GFP+/GFP− ratio by longitudinal flow cytometric analysis as a measure of the negative impact of the RNAi treatment on the cellular proliferation rate. We show that this assay is sensitive, easy to use and internally controlled for assessing subtle effects on cell proliferation of lentiviral transduction and transgene expression.

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Acknowledgements

This research was supported by the Dutch AIDS fund (grant 2006006 and 2007028). We thank Berend Hooibrink for FACS sorting, Stephan Heynen for CA-p24 enzyme-linked immunosorbent assay experiments and Renée van der Sluis and Dave Speijer for useful discussions. We also thank the Belgian Federal Government for financial support through the Inter-University Attraction Pole grant P6/41.

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Authors and Affiliations

  1. Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

    J J M Eekels, A O Pasternak, A M Schut, R E Jeeninga & B Berkhout

  2. Department of Human Genetics, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

    D Geerts

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  1. J J M Eekels
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  2. A O Pasternak
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  3. A M Schut
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  4. D Geerts
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  5. R E Jeeninga
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  6. B Berkhout
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Correspondence to B Berkhout.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on Gene Therapy website

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Eekels, J., Pasternak, A., Schut, A. et al. A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation. Gene Ther 19, 1058–1064 (2012). https://doi.org/10.1038/gt.2011.191

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