Abstract

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance1,2 that can confound analyses and undermine reproducibility3. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

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Acknowledgements

We thank all members of our laboratories, IMBA/IMP and VBCF services for support and Life Science Editors for assistance; B. Knapp, I. Filipuzzi and T. Aust for clone picking, N. R. Movva and T. Bouwmeester (NIBR) for support, and K. Handler for the differentiation protocols. The Haplobank is funded by the Austrian National Bank (OeNB), an Advanced ERC grant and Era of Hope/National Coalition against Breast Cancer/DoD (to J.M.P.). U.E. is a Wittgenstein Prize fellow. D.B. is supported by FWF P23308-B13. A.S. is supported by an ERC Consolidator Grant, Boehringer Ingelheim and FFG.

Author information

Author notes

    • Ulrich Elling
    •  & Reiner A. Wimmer

    These authors contributed equally to this work.

Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria

    • Ulrich Elling
    • , Reiner A. Wimmer
    • , Andreas Leibbrandt
    • , Thomas Burkard
    • , Georg Michlits
    • , Alexandra Leopoldi
    • , Dana Abdeen
    • , Sergei Zhuk
    • , Cornelia Handl
    • , Julia Liebergesell
    • , Maria Hubmann
    • , Anna-Maria Husa
    • , Manuela Kinzer
    • , Nicole Schuller
    • , Ellen Wetzel
    • , Nina van de Loo
    • , Jorge Arturo Zepeda Martinez
    • , Chukwuma A. Agu
    • , Oliver Bell
    •  & Josef M. Penninger
  2. Vienna Biocenter Core Facilities, Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria

    • Thomas Micheler
  3. MRC Laboratory for Molecular Cell Biology and Institute for the Physics of Living Systems, University College London, London, UK

    • Irene M. Aspalter
  4. Novartis Institutes for BioMedical Research, Basel, Switzerland

    • David Estoppey
    • , Ralph Riedl
    •  & Dominic Hoepfner
  5. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    • Fengtang Yang
    •  & Beiyuan Fu
  6. Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr Gasse 9, Vienna, Austria

    • Thomas Dechat
    •  & Dieter Blaas
  7. Paul Ehrlich Institut, Paul Ehrlich Strasse 51–59, 63225 Langen, Germany

    • Zoltán Ivics
  8. Max-Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

    • Holger Gerhardt
  9. German Center for Cardiovascular Research, Berlin, Germany

    • Holger Gerhardt
  10. Berlin Institute of Health, Berlin, Germany

    • Holger Gerhardt
  11. Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Dr. Bohr Gasse 7, 1030 Vienna, Austria

    • Alexander Stark

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Contributions

U.E. generated the haploid library with technical support from A.Lei., C.H., J.L., M.H., A.-M.H., M.K., N.S., E.W., N.v.d.L., D.H., R.R. and D.E. U.E., R.A.W. and A.Leo. characterized cell lines. A.Lei., G.M., U.E., D.B. and T.D. performed rhinovirus work. A.S., T.B. and T.M. wrote the bioinformatics algorithms and set up the Haplobank website. S.Z. performed RACE experiments, F.Y. and B.F. performed karyotyping experiments and C.A.A. supported standardization. J.A.Z.M. and O.B. performed ATAC-sequencing. Z.I. advized on mutagenesis vectors. R.A.W., I.M.A., D.A., A.Leo. and H.G. performed blood vessel experiments. U.E. and J.M.P. coordinated the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ulrich Elling or Josef M. Penninger.

Reviewer Information Nature thanks S. Narumiya and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Reporting Summary

  2. 2.

    Supplementary Figures

    This file contains full uncropped scans of DNA gels and Western blots used in Extended Data Figure 8.

  3. 3.

    Supplementary Table

    This table shows the numbers of clones available with respect to different mutagens, orientation of the inserted gene trap to gene transcription, as well as the number of different genes hit. A gene is defined as the genomic region between the transcriptional start and stop sites. www.haplobank.at

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DOI

https://doi.org/10.1038/nature24027

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