1. Identification of Genes That Modulate Susceptibility to Formaldehyde and Imatinib by Functional Genomic Screening in Human Haploid KBM7 Cells
- Author
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Cliona M. McHale, Luoping Zhang, Martyn T. Smith, Hua Shen, Cham Jung, Syed I. Haider, and Susie Zhang
- Subjects
0301 basic medicine ,Mutant ,Drug Resistance ,Haploidy ,Toxicology ,0302 clinical medicine ,RNA interference ,Chronic ,KBM7 ,Cancer ,Leukemic ,Genetics ,Gene knockdown ,Leukemia ,Gene Expression Regulation, Leukemic ,Myeloid leukemia ,Hematology ,Pharmacology and Pharmaceutical Sciences ,Corrigenda ,haploid ,5.1 Pharmaceuticals ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,Imatinib Mesylate ,RNA Interference ,Drug ,Development of treatments and therapeutic interventions ,functional genomics ,Functional genomics ,Biotechnology ,Cell Survival ,Antineoplastic Agents ,Biology ,Functional Genomic Screening in a Human Haploid Cell Line ,Transfection ,Cell Line ,Dose-Response Relationship ,03 medical and health sciences ,Rare Diseases ,Formaldehyde ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Humans ,Gene ,imatinib ,Gene knockout ,Cell Proliferation ,Dose-Response Relationship, Drug ,Prevention ,Human Genome ,High-Throughput Screening Assays ,Good Health and Well Being ,030104 developmental biology ,Imatinib mesylate ,Gene Expression Regulation ,Drug Resistance, Neoplasm ,Mutation ,Neoplasm ,BCR-ABL Positive ,Myelogenous - Abstract
Though current functional genomic screening systems are useful for investigating human susceptibility to chemical toxicity, they have limitations. Well-established, high-throughput yeast mutant screens identify only evolutionarily conserved processes. RNA interference can be applied in human cells but is limited by incomplete gene knockout and off-target effects. Human haploid cell screening is advantageous as it requires knockdown of only a single copy of each gene. A human haploid cell mutant library (KBM7-Mu), derived from a chronic myeloid leukemia (CML) patient, was recently developed and has been used to identify genes that modulate sensitivity to infectious agents and pharmaceutical drugs. Here, we sought to improve the KBM7-Mu screening process to enable efficient screening of environmental chemicals. We developed a semi-solid medium based screening approach that cultures individual mutant colonies from chemically resistant cells, faster (by 2–3 weeks) and with less labor than the original liquid medium-based approach. As proof of principle, we identified genetic mutants that confer resistance to the carcinogen formaldehyde (FA, 12 genes, 18 hits) and the CML chemotherapeutic agent imatinib (6 genes, 13 hits). Validation experiments conducted on KBM7 mutants lacking each of the 18 genes confirmed resistance of 6 FA mutants (CTC1, FCRLA, GOT1, LPR5, M1AP, and MAP2K5) and 1 imatinib-resistant mutant (LYRM9). Despite the improvements to the method, it remains technically challenging to limit false positive findings. Nonetheless, our findings demonstrate the broad applicability of this optimized haploid approach to screen toxic chemicals to identify novel susceptibility genes and gain insight into potential mechanisms of toxicity.
- Published
- 2016