Letter | Published:

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing

Nature Biotechnology volume 29, pages 542546 (2011) | Download Citation

Abstract

Insertional mutagenesis in a haploid background can disrupt gene function1. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones1 enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites2. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.

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Acknowledgements

We would like to thank D. Sabatini and J. Roix for critical reading of this manuscript, S. Boulant, M. Nibert and R. Rooswinkel for providing reagents, and T. DiCesare for graphics support. I.W. was supported by a PhD fellowship from the Boehringer Ingelheim Fonds. T.R.B. was supported by US National Institutes of Health grant R21-HG004938-01. The construct for the full operon of E. coli CDT was generously provided by J. Kaper, University of Maryland School of Medicine, Baltimore.

Author information

Author notes

    • Jan E Carette
    • , Vincent A Blomen
    •  & Thijn R Brummelkamp

    Present addresses: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA (J.E.C.) and The Netherlands Cancer Institute, Amsterdam, The Netherlands (V.A.B. and T.R.B.).

    • Jan E Carette
    • , Carla P Guimaraes
    • , Irene Wuethrich
    •  & Vincent A Blomen

    These authors contributed equally to this work.

Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Jan E Carette
    • , Carla P Guimaraes
    • , Irene Wuethrich
    • , Vincent A Blomen
    • , Malini Varadarajan
    • , Chong Sun
    • , George Bell
    • , Bingbing Yuan
    • , Hidde L Ploegh
    •  & Thijn R Brummelkamp
  2. Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

    • Markus K Muellner
    •  & Sebastian M Nijman
  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Hidde L Ploegh

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Contributions

J.E.C., C.P.G., I.W., V.A.B., M.V., C.S., M.K.M., S.M.N. and T.R.B. designed and performed experiments. B.Y., G.B., J.E.C., V.A.B. and T.R.B. were involved in the bioinformatics. J.E.C., C.P.G., I.W., V.A.B., H.L.P. and T.R.B. wrote the manuscript.

Competing interests

J.E.C. and T.R.B. are named inventors on a patent application on technology described in this manuscript. S.M.N. and T.R.B. are co-founders of an early-stage startup company involved in haploid genetic approaches.

Corresponding authors

Correspondence to Hidde L Ploegh or Thijn R Brummelkamp.

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DOI

https://doi.org/10.1038/nbt.1857

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