title: Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study.
abstract: BACKGROUND
  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 μM Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48h 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.
  
  
  CONCLUSIONS
  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.
authors: Department of Epidemiology and Environmental Health, The State University of New York at Buffalo, United States; School of Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, PR China.
authors: Jinqiu
authors: J
authors: Zhu
authors: Department of Epidemiology and Environmental Health, The State University of New York at Buffalo, United States; Department of Pharmacology and Toxicology, The State University of New York at Buffalo, United States.
authors: Amber
authors: A
authors: Dubois
authors: Department of Epidemiology and Environmental Health, The State University of New York at Buffalo, United States.
authors: Yichen
authors: Y
authors: Ge
authors: Department of Epidemiology and Environmental Health, The State University of New York at Buffalo, United States; Department of Pharmacology and Toxicology, The State University of New York at Buffalo, United States.
authors: James A
authors: JA
authors: Olson
authors: Department of Epidemiology and Environmental Health, The State University of New York at Buffalo, United States; Department of Pharmacology and Toxicology, The State University of New York at Buffalo, United States. Electronic address: xuefengr@buffalo.edu.
authors: Xuefeng
authors: X
authors: Ren
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004390:Chlorpyrifos / Q000633:toxicity*; D004351:Drug Resistance / Q000187:drug effects* / Q000235:genetics; D054750:Ecotoxicology; D004785:Environmental Pollutants / Q000633:toxicity*; D005820:Genetic Testing / Q000379:methods*; D006238:Haploidy; D006801:Humans; D008957:Models, Genetic; D060888:Real-Time Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D004785:Environmental Pollutants; D004390:Chlorpyrifos
keywords: Chlorpyrifos; KBM7-mutated haploid cells; Loss-of-function genetic screening; Susceptibility to environmental chemical exposure