Transgenic RNA interference in ES cell–derived embryos recapitulates a genetic null phenotype (original) (raw)

Nature Biotechnology volume 21, pages 559–561 (2003)Cite this article

Abstract

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo1. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms2,3. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing4,5. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA6,7,8. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method9. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation10.

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Acknowledgements

We thank Sue MacMaster and Lois Byers for tetraploid aggregations and uterine transfers and Andras Nagy for critical discussions. This research was supported by the Canadian Institutes of Health Research and a Terry Fox Programme Project grant from the National Cancer Institute of Canada.

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Author notes

  1. Tilo Kunath and Gerald Gish: These authors contributed equally to this study.

Authors and Affiliations

  1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, ON, Canada
    Tilo Kunath, Gerald Gish, Heiko Lickert, Nina Jones, Tony Pawson & Janet Rossant
  2. Department of Molecular and Medical Genetics University of Toronto, University of Toronto, Toronto, M5S 1A8, ON, Canada
    Tilo Kunath, Tony Pawson & Janet Rossant

Authors

  1. Tilo Kunath
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  2. Gerald Gish
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  3. Heiko Lickert
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  4. Nina Jones
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  5. Tony Pawson
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  6. Janet Rossant
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Correspondence toTony Pawson or Janet Rossant.

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The authors declare no competing financial interests.

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Kunath, T., Gish, G., Lickert, H. et al. Transgenic RNA interference in ES cell–derived embryos recapitulates a genetic null phenotype.Nat Biotechnol 21, 559–561 (2003). https://doi.org/10.1038/nbt813

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