Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 429, Issue 18, Pages 2780-2789Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2017.07.020
Keywords
Gene trap; piggyBac transposon; RNAseq; haploid mammalian cells; genetic screenings
Categories
Funding
- Spanish Ministry of Economy and Competitivenes [SAF2014-59498-R, SAF2014-57791-REDC]
- Fundacion Botin, Banco Santander, through its Santander Universities Global Division
- Fundacio La Marato de TV3
- Howard Hughes Medical Institute
- European Research Council [ERC-617840]
- La Caixa Foundation
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The development of haploid mammalian cell lines, coupled to next-generation sequencing, has recently facilitated forward genetic screenings in mammals. For mutagenesis, retrovirus- or transposon-based gene traps are frequently used. Current methods to map gene-trap insertions are based on inverse or splinkerette PCR, which despite their efficacy are prone to artifacts and do not provide information on expression of the targeted gene. Here, we describe a new RNA sequencing-based method (Trap(seq)) to map gene-trap insertions. By recognizing chimeric mRNAs containing gene-trap sequences spliced to an exon, our method identifies insertions that lead to productive trapping. When applied to individual mutant clones, our method provides a fast and cost-effective way that not only identifies the insertion site but also reveals its impact on the expression of the trapped gene. As proof of principle, we conducted two independent screenings for resistance against 6-thioguanine and an ATR inhibitor, which identified mutations known to provide resistance to these reagents and revealed ECT2 as a novel determinant for the sensitivity to ATR inhibition. (C) 2017 Elsevier Ltd. All rights reserved.
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