Investigating the molecular causes of inherited thrombocytopenia using CRISPR-Cas9 gene editing and super-resolution microscopy

[摘要] Inherited thrombocytopenias (ITs) are a heterogenous group of disorders characterised by an abnormal platelet count (<150 x 109/L). Functional investigations into the mechanistic causes of ITs often suffer from numerous technical limitations, including a lack of patient material and the absence of platelet producing cell lines. In this thesis, protocols to address these issues through the application of CRISPR (Clustered Regularly Interspaced Palindromic Repeats) genome editing and stem cell differentiation are developed and applied to the study of the platelet and megakaryocyte specific β-tubulin isoform encoded by the TubB1 gene. First, a novel method of single molecule super-resolution microscopy through the CRISPR mediated editing of cell lines is devised, and attempts to transfer this approach to stem cells are currently underway. Secondly, existing methods for the differentiation of induced pluripotent stem cells into megakaryocytes are adapted to generate mature, proplatelet forming cells for in vitro phenotypic screening. Finally, two families from the UK GAPP (Genotyping and Phenotyping of Platelets) study with TubB1 mutations affecting C-terminal function are investigated to determine the role of C-terminal post-translational modifications (PTMs) on ITs. Two novel PTMs (polyglutamylation and polyglycylation) which regulate motor protein processivity in both platelet production and function are reported.

[发布机构] University:University of Birmingham;Department:Institute of Cardiovascular Sciences

[关键词] Q Science;QH Natural history;QH426 Genetics