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Cytopenia induction by 5-fluorouracil identifies thrombopoietic mutants in sensitized ENU mutagenesis screens |
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| Authors: | Anderson Nicole M Berberovic Zorana Berndl Elizabeth Bailey Monica L Flenniken Ann M Osborne Lucy R Adamson S Lee Rossant Janet Wang Chen Minden Mark D McNagny Kelly M Paulson Robert F Barber Dwayne L Stanford William L |
| Affiliation: | a Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ont, Canada b Institute of Biomaterials and Biomedical Engineering, Faculty of Medicine, University of Toronto, Toronto, Ont., Canada c Department of Medicine, Faculty of Medicine, University of Toronto, Toronto, Ont., Canada d Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toronto, Toronto, Ont., Canada e Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ont., Canada f Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, Ont., Canada g Samuel Lunenfeld Research Institute, Toronto, ON, Canada h Mount Sinai Hospital, Toronto, Ont., Canada i Toronto Centre for Phenogenomics, Toronto, Ont., Canada j Hospital for Sick Children, Toronto, Ont., Canada k The Biomedical Research Centre, University of British Columbia, Vancouver, BC., Canada l Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pa., USA m Campbell Family Cancer Research Institute, Ontario Cancer Institute, Toronto, Ont., Canada n Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, Ont., Canada |
| Abstract: | The ability of random mutagenesis techniques to annotate the mammalian genome can be hampered due to genetic redundancy and compensatory pathways that mask heterozygous mutations under homeostatic conditions. The objective of this study was to devise a pharmacologically sensitized screen using the chemotherapeutic drug, 5-fluorouracil (5FU), to induce cytopenia. 5FU dose was optimized in the 129/SvImJ, C57BL/6J, BALB/cJ, and C3H/HeJ strains of laboratory mice. N-ethyl-N-nitrosourea (ENU) mutagenesis was performed on 129/SvImJ males and phenotypic variants were identified by backcrossing on to the C57BL/6J background. G1 animals were challenged with 100 μg/g 5FU and phenodeviants with altered platelet recovery were monitored. Of 546 G1 animals tested, 15 phenodeviants were identified that displayed increased baseline platelet number, a platelet overshoot, or delayed platelet recovery, thereby demonstrating the utility of this approach for uncovering mutations in megakaryocyte and platelet development. Four G1 mice were selected for further analysis. The phenotypes were heritable in all four strains and genetic mapping identified a chromosome location in two of the three G2 lines tested. In conclusion, our group has developed a sensitized random mutagenesis screen utilizing 5FU and has shown that the strain combination of 129/SvImJ × C57BL/6J is robust for identification of founder lines with defects in megakaryocyte and platelet development. |
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