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A reversible gene trap collection empowers haploid genetics in human cells

Nature Methods volume 10, pages 965–971 (2013)Cite this article

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Abstract

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-β, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

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Figure 1: Genomic and proteomic characterization of KBM7 cells.
Figure 2: A pipeline for the generation of haploid 'gene-trapped' cells.
Figure 3: Gene-trapped mutants are reversible and can resemble gene knockouts.
Figure 4: Molecular portraits of mutant KBM7 cells establish genotype-phenotype relationships.

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Acknowledgements

We thank G. Winter and members of the Brummelkamp, Nijman and Superti-Furga laboratories for discussions and technical assistance, R. Martins for help with the illustrations in Figure 2, O. Majdic (Medical University of Vienna) for providing antibodies, J. Carette for advice on gene trap vector design, and H. Pickersgill for manuscript editing and suggestions. C. Banning was supported by a FemPower grant from Zentrum für Innovation und Technologie (Die Technologieagentur der Stadt Wien), and A.L. and M.L. were supported by a Zentrum für Innovation und Technologie Life Sciences 2011 grant. M.R. was supported by European Molecular Biology Organization fellowship (ALTF1346-2011). K.P. was supported by a European Research Council grant (ERC-2009-AdG-250179-i-FIVE).

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Authors and Affiliations

  1. Haplogen GmbH, Vienna, Austria

    Tilmann Bürckstümmer, Carina Banning, Philipp Hainzl, Richard Schobesberger, Ana Lapao, Melissa Liszt, Benjamin Eizinger, Wolfgang Fischl, Sejla Salic, M Rita Taba Casari & Georg Casari

  2. Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria

    Philipp Hainzl, Claudia Kerzendorfer, Florian M Pauler, Doris Chen, Nicole Them, Fiorella Schischlik, Manuele Rebsamen, Michal Smida, Ferran Fece de la Cruz, Ana Lapao, Melissa Liszt, Philipp M Guenzl, Tomasz Konopka, Bianca Gapp, Katja Parapatics, Barbara Maier, Sylvia Knapp, Keiryn L Bennett, Christoph Bock, Jacques Colinge, Robert Kralovics, Thijn R Brummelkamp, Giulio Superti-Furga & Sebastian M B Nijman

  3. The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Vincent A Blomen & Thijn R Brummelkamp

  4. Department of Medicine 1, Laboratory of Infection Biology, Medical University of Vienna, Vienna, Austria

    Barbara Maier & Sylvia Knapp

  5. Institute of Immunology, Medical University of Vienna, Austria

    Johannes Stöckl

  6. Max F. Perutz Laboratories, University of Vienna, Vienna, Austria

    Gustav Ammerer

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  1. Tilmann Bürckstümmer
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Contributions

S.M.B.N., T.R.B. and G.S.-F. conceived the haploid gene-trap mutant collection and provided overall guidance. S.M.B.N. and T.B. analyzed data and, together with G.S.-F. and T.R.B., wrote the paper. S.M.B.N., T.R.B., T.B. and G.C. conceived the gene-trap vector design including barcodes and loxP sites and the clone-mapping pipeline. T.B. and C. Banning supervised the establishment of the mutant collection and performed validation experiments. P.H., A.L., M.L., W.F., S.S. and M.R.T.C. assisted in the establishment of the mutant collection and validation experiments. F.M.P., D.C., N.T., F.S., B.E., P.M.G., V.A.B., T.K., B.G., C. Bock and R.K. generated the samples for DNA and RNA sequencing and SNP arrays, and analyzed the data. R.S., B.E. and G.C. established the clone-mapping bioinformatics pipeline and databases. C.K., M.R., M.S. and F.F.d.l.C. performed clone-validation experiments. K.P., K.L.B. and J.C. generated samples for mass spectrometry and analyzed the data. B.M., J.S. and S.K. performed and analyzed the leukocyte typing experiments. G.C. and G.A. assisted in platform design.

Corresponding authors

Correspondence to Tilmann Bürckstümmer, Thijn R Brummelkamp, Giulio Superti-Furga or Sebastian M B Nijman.

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Competing interests

G.A., G.C., T.R.B., G.S.-F. and S.M.B.N. are founders and shareholders of Haplogen. T.B., C.Banning, P.H., A.L., M.L., W.F., S.S. and M.R.T.C. are employees of Haplogen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1, 2, 7, 9 and 11–14 (PDF 7324 kb)

Supplementary Table 3

Variants from exome and whole genome (100-bp paired-end) sequencing. (XLSX 23712 kb)

Supplementary Table 4

Comprehensive proteome profiling. (XLSX 1742 kb)

Supplementary Table 5

mRNA and protein expression. (XLSX 426 kb)

Supplementary Table 6

KEGG pathway analysis of gene trap collection. (XLSX 21 kb)

Supplementary Table 8

KEGG pathway coverage of clone collection. (XLS 53 kb)

Supplementary Table 10

Detailed information concerning all available gene trap clones. (XLS 1802 kb)

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Bürckstümmer, T., Banning, C., Hainzl, P. et al. A reversible gene trap collection empowers haploid genetics in human cells. Nat Methods 10, 965–971 (2013). https://doi.org/10.1038/nmeth.2609

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