文献类型：期刊文献

英文题名：Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study

作者：Zhu, Jinqiu Dubois, Amber Ge, Yichen Olson, James A. Ren, Xuefeng

第一作者：朱金秋;Zhu, Jinqiu

通信作者：Ren, XF[1]

机构：[1]SUNY Buffalo, Dept Epidemiol & Environm Hlth, Buffalo, NY 14221 USA;[2]SUNY Buffalo, Dept Pharmacol & Toxicol, Buffalo, NY 14221 USA;[3]Guizhou Inst Technol, Sch Pharmaceut Engn, Guiyang 550003, Peoples R China

第一机构：SUNY Buffalo, Dept Epidemiol & Environm Hlth, Buffalo, NY 14221 USA

通信机构：corresponding author), SUNY Buffalo, 276 Farber Hall, Buffalo, NY 14221 USA.

年份：2015

卷号：76

起止页码：76-82

外文期刊名：JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS

收录：;Scopus(收录号：2-s2.0-84941275704);WOS:【SCI-EXPANDED(收录号:WOS:000365103400011)】；

基金：This work was supported by a start-up fund (to X.R.) provided by University at Buffalo. Support was also provided by NIEHS grant R21ES022329 and R01ES022629 to X.R.

语种：英文

外文关键词：Chlorpyrifos; KBM7-mutated haploid cells; Loss-of-function genetic screening; Susceptibility to environmental chemical exposure

摘要：Introduction: High-throughput loss-of-function genetic screening tools in yeast or other model systems except in mammalian cells have been implemented to study human susceptibility to chemical toxicity. Here, we employed a newly developed human haploid cell (KBM7)-based mutagenic screening model (KBM7-mu cells) and examined its applicability in identifying genes whose absence allows cells to survive and proliferate in the presence of chemicals. Methods: KBM7-mu cells were exposed to 200 mu M Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48 h of treatment. After a 2-3 week period of continuous CPF exposure, survived single cell colonies were recovered and used for further analysis. DNA isolated from these cells was amplified using Splinkerette PCR with specific designed primers, and sequenced to determine the genomic locations with virus insertion and identify genes affected by the insertion. Quantitative realtime reverse transcription PCR (qRT-PCR) was used to confirm the knockdown of transcription of identified target genes. \ Results: We identified total 9 human genes in which the cells carrying these genes conferred the resistance to CPF, including AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, MZF1-AS1 and PPTC7. MZF1-AS1 is an antisense RNA and not included in the further analysis. qRT-PCR results showed that the expression of 6 genes was either significantly reduced or completely lost. There were no changes in the expression of DCAF12 and AGPAT6 genes between the KBM7-mu and the control KBM7 cells. Discussion: The KBM7-mu genetic screening system can be modified and applied to identify novel susceptibility genes in response to environmental toxicants, which could provide valuable insights into potential mechanisms of toxicity. (C) 2015 Elsevier Inc. All rights reserved.