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dc.contributor.authorBeutler, Lucinda
dc.date.accessioned2019-05-31
dc.date.available2019-05-31
dc.date.issued2018-12-31
dc.identifier.urihttp://hdl.handle.net/2123/20481
dc.description.abstractGFI1B is a transcriptional repressor that is a major regulator of megakaryocytic and erythroid differentiation and development. The cause of a bleeding disorder in a four- generation Australian family, with cell changes consistent with aberrant platelet structure and function, and red blood cell morphology, showing autosomal dominant inheritance, was identified as being caused by a single nucleotide insertion into the GFI1B gene (c.880_881insC). The aims of this thesis were to study the effects of this mutant GFI1B on erythrocyte development in a cell culture model; to study how loss of Gfi1b function affects megakaryocytic development in a conditional knockout mouse model; and to examine the effects of human GFI1Bc.880_881insC on megakaryocytic / erythroid cell development in a transgenic mouse model, with correlation to the human phenotype. Separate lines of erythroleukaemic K562 cells were transfected with GFI1BWT and GFI1Bc.880_881insC for studies into red cell formation. Inconclusive results from constant loss of transfection led to the view that a cell model derived from iPSC from affected family members would provide a more stable system for this study. For the conditional knockout mouse model, C57BL/6J mice with floxed Gfi1b alleles were cross-bred to transgenic mice expressing Cre recombinase under the platelet factor 4 (Pf4) promoter to create a null allele. Mice with retained floxed alleles were controls. The effects of the loss of ­Gfi1b along the megakaryocytic lineage included 60-fold reduction in platelet count; aberrant megakaryocyte morphology, with high DNA ploidy and increased numbers in bone marrow and spleen; high plasma TPO level; enlarged spleens with evidence of stress erythropoiesis; and reduced red cell indices. Reported studies in the literature corroborated these findings. C57BL/6J transgenic mice with either GFI1BWT or GFI1Bc.880_881insC with a lox-STOP-lox cassette inserted at the collagen Col1a1 locus were crossed with transgenic mice expressing Pf4-Cre for the transgenic mouse model. The premise, that the human mutant GFI1B would cause a dominant negative effect on the endogenous mouse Gfi1b, was not seen in the lack of a bleeding phenotype, and evidence of normal haematological parameters in these mice. A knock-in model with GFI1Bc.880_881insC replacing Gfi1b may be more effective.en_AU
dc.publisherUniversity of Sydneyen_AU
dc.publisherSydney Medical Schoolen_AU
dc.publisherNorthern Clinical Schoolen_AU
dc.rightsThe author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.en_AU
dc.subjectGFI1B, platelets; conditional knockout; bleeding disorderen_AU
dc.titlePlatelet and erythrocyte functional defects caused by mutations of GFI1Ben_AU
dc.typePhD Doctorateen_AU
dc.type.pubtypeDoctor of Philosophy Ph.D.en_AU


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