Trap seq: An RNA Sequencing-Based Pipeline for the Identification of Gene-Trap Insertions in Mammalian Cells

JOURNAL OF MOLECULAR BIOLOGY

卷:429

Article

Mayor-Ruiz, Cristina¹  Dominguez, Orlando²  Fernandez-Capetillo, Oscar^1,3 

[1] Spanish Natl Canc Res Ctr, Genom Instabil Grp, Madrid 28029, Spain

[2] Spanish Natl Canc Res Ctr, Genom Unit, Madrid 28029, Spain

[3] Karolinska Inst, Div Genome Biol, Dept Med Biochem & Biophys, Sci Life Lab, S-17121 Stockholm, Sweden

关键词: Gene trap;    piggyBac transposon;    RNAseq;    haploid mammalian cells;    genetic screenings;   

DOI  :  10.1016/j.jmb.2017.07.020

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

Free