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. 2017 Mar 1:4:170020.
doi: 10.1038/sdata.2017.20.

Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells

Stephen J Pettitt  1   2 , Dragomir B Krastev  1   2 , Helen N Pemberton  1   2 , Yari Fontebasso  1   2 , Jessica Frankum  1   2 , Farah L Rehman  1   2 , Rachel Brough  1   2 , Feifei Song  1   2 , Ilirjana Bajrami  1   2 , Rumana Rafiq  1   2 , Fredrik Wallberg  2 , Iwanka Kozarewa  3 , Kerry Fenwick  3 , Javier Armisen-Garrido  3 , Amanda Swain  3 , Aditi Gulati  1   2 , James Campbell  1   2 , Alan Ashworth  1   2 , Christopher J Lord  1   2
Affiliations

Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells

Stephen J Pettitt et al. Sci Data. .

Abstract

We describe a screen for cellular response to drugs that makes use of haploid embryonic stem cells. We generated ten libraries of mutants with piggyBac gene trap transposon integrations, totalling approximately 100,000 mutant clones. Random barcode sequences were inserted into the transposon vector to allow the number of cells bearing each insertion to be measured by amplifying and sequencing the barcodes. These barcodes were associated with their integration sites by inverse PCR. We exposed these libraries to commonly used cancer drugs and profiled changes in barcode abundance by Ion Torrent sequencing in order to identify mutations that conferred sensitivity. Drugs tested included conventional chemotherapeutics as well as targeted inhibitors of topoisomerases, poly(ADP-ribose) polymerase (PARP), Hsp90 and WEE1.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Mutagenesis and screening strategy.
(a) Screen setup. (b) Structure of mutagenic transposon, showing gene trap splice acceptor construct and random barcode regions (N25). (c) Inverse PCR strategy to associate barcodes with integration sites.
Figure 2
Figure 2. Genomic coverage of libraries.
(a) Cumulative number of unique genes disrupted in libraries 1–10. (b) Breakdown of genes disrupted by expression in mouse embryonic stem cells, The fraction of genes in each category with at least one integration is plotted. Single cell RNA-seq expression data and categorisation have been previously published.
Figure 3
Figure 3. Technical validation.
(a) Concordance of read counts in two replicate DMSO-treated samples. (b) Schematic of spiking experiment. (c) Volcano plot of spiking experiment results. P values (exact test) and fold changes were calculated using edgeR. The point corresponding to the barcode in the spiked Brca2 mutant is labelled. (d) Volcano plot of barcodes corresponding to Ewsr1 integrations in all PB3 libraries exposed to the PARP inhibitor BMN 673. (e) Validation of Ewsr1 hit. CRISPR knockout ES cells (L5.11) are more sensitive to BMN 673 than parental JM8A3 cells. Surviving fraction is measured using CellTiterGlo after five days growth in the indicated drug concentration; mean and s.d. of five replicates is plotted. (f) Western blot demonstrating knockout of Ewsr1 in CRISPR mutant. (g,h) Knockdown of the human homologue, EWSR1, in MCF7 cells using shRNA causes BMN 673 sensitivity.
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References

Data Citations

    1. Pettitt S. J. 2016. Figshare. https://dx.doi.org/10.6084/m9.figshare.3469157.v1 - DOI
    1. Pettitt S. J. 2016. Figshare. https://dx.doi.org/10.6084/m9.figshare.3469112.v1 - DOI
    1. Pettitt S. J. 2016. Figshare. https://dx.doi.org/10.6084/m9.figshare.4213170.v3 - DOI

References

    1. Carette J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009). - PubMed
    1. Carette J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343 (2011). - PMC - PubMed
    1. Elling U. et al. Forward and Reverse Genetics through Derivation of Haploid Mouse Embryonic Stem Cells. Cell Stem Cell 9, 563–574 (2011). - PMC - PubMed
    1. Leeb M. & Wutz A. Derivation of haploid embryonic stem cells from mouse embryos. Nature 479, 131–134 (2011). - PMC - PubMed
    1. Reiling J. H. et al. A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin. Proc. Natl. Acad. Sci. USA 108, 11756–11765 (2011). - PMC - PubMed

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