Novel KBM7 human haploid cell based mutagenized genetic screening model and its application in identifying human susceptibility genes to toxic chemicals exposure: Chlorpyrifos as a case study
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Genetic screening models, such as those in yeast and RNA interference, have become very popular tools for phenotype selection. Furthermore, genetic screening systems in non-mammalian models have successfully been used to study human susceptibility to chemical exposure, as well as deciphering mechanisms of action. Mammalian cell lines are limited as tools for genetic analysis due to their diploid karyotype. A newly developed human haploid cell based mutagenic loss-of-functional genetic screening model was recently described. The mutagenic model was developed from the KBM7 human near-haploid cell line, guaranteeing loss of gene function with viral insertion. The aim of this study was to establish and optimize the protocol of this model and explore its application in identifying human susceptibility genes to toxic chemicals exposure. Chlorpyrifos (CPF), an organophosphate (OP) pesticide, is one of the most widely used OPs despite its known ability to cause neurotoxicity. The most common and extensively known mechanism CPF toxicity is through the inhibition of cholinesterase activity. However, various other neurobehavioral and developmental deficits, unrelated to cholinesterase inhibition, have been associated with CPF exposure. The mechanisms behind these effects are not fully known and understood. Here, we applied this human haploid cell based Mutagenized genetic screening model to study the genetic susceptibility to CPF exposure. KBM7-mutated cells were treated with 200 μM CPF, a dose causing approximately 40-65% death of cells after 48 hours of treatment. After a 2-3 week period of treatment, cells were identified which carry genes with deficient functions resulting in resistance to CPF-induced toxicity. Approximately 50 CPF resistant cell colonies were recovered. DNA fragments from the gene mutated by viral insertion were amplified for sequencing and gene identification. Genes AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, LOC100131691, and PPTC7 were identified as playing a potential role in CPF toxicity, due to their ability to impart a resistant phenotype when disrupted. Furthermore, the results indicate that the KBM7-mutagenic loss-of-function model can be used to identify novel susceptibility genes to environmental toxins, such as CPF, as well as to gain insight into potential mechanisms of toxicity.