The CRUK Gene Function Laboratory, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK.
Breast Cancer Now Research Centre, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK.
Sci Data. 2017 Mar 1;4:170020. doi: 10.1038/sdata.2017.20.
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.
我们描述了一种利用单倍体胚胎干细胞检测细胞对药物反应的筛选方法。我们利用 piggyBac 基因捕获转座子整合生成了十个突变文库，总共包含约 100000 个突变克隆。随机条形码序列被插入转座子载体中，通过扩增和测序条形码来测量携带每个插入的细胞数量。通过反向 PCR 将这些条形码与它们的整合位点相关联。我们将这些文库暴露于常用的癌症药物中，并通过 Ion Torrent 测序来分析条形码丰度的变化，以鉴定赋予药物敏感性的突变。测试的药物包括传统的化疗药物以及拓扑异构酶、聚（ADP-核糖）聚合酶（PARP）、Hsp90 和 WEE1 的靶向抑制剂。
