Journal
EXPERIMENTAL HEMATOLOGY
Volume 39, Issue 3, Pages 305-320Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.exphem.2010.12.009
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Funding
- National Institutes of Health (Bethesda, MD, USA) [HL088468]
- CIHR [FRN 74611, MOP-93580]
- Heart and Stroke Foundation of Ontario [NA-6363]
- Canada Research Chair
- Dina Gordon Malkin Ontario Graduate Scholarship in Science Technology
- Heart & Stroke/Richard Lewar Centre of Excellence
- Canadian Stem Cell Network
- Heart & Stroke Foundation of Canada (HSFC)
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Objective. Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS. Materials and Methods. We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels. Results. Although Ank1(E924X/E924X): red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1. Conclusions. The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS. (C) 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
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