Browsing by Author "HIAM OSAMA HASSAN MULLA"

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Analysis of erythropoiesis in BEL–A2 cells with a CRISPR–Cas9 edited KIT gene
HIAM OSAMA HASSAN MULLA; Dr Belinda Singleton
The BEL-A2 cell line is an immortalized erythroid cell line created in Bristol by lentiviral transduction (HPV16-E6/E7 construct) of adult bone marrow-derived CD34+ cells. The erythroid cell line was created in hope that in the future, such cell line would provide NHSBT with sustainable supply of in-vitro produced red blood cells (RBCs) for diagnostic and therapeutic purposes. However, growing BEL-A2 cells depends completely on the addition of two important cytokines in tissue culture medium: erythropoietin (EPO) and stem cell factor (SCF). In humans, activating mutations in the KIT gene (encoding SCF-ligand) can result in SCF-independent activity. Thus, CRISPR-Cas9 technology was used to edit the KIT gene in BEL-A2 cell line in aim to produce a phenotype capable of cellular expansion in SCF-free environment. The main aim of this project is to observe whether the CRISPR-Cas9 edited cells (KIT-4 cell line) will remain capable of terminal erythroid maturation and compare them to the original BEL-A2 cells. This will be done through assessing the growth characteristics and proliferation capacity of both lines through cell counts and doubling time calculations. The morphological features will be determined using cytospins and microscopy, whereas flow cytometry analysis will be used to measure proliferative capacity. Determining the molecular nature of KIT-4 cell line sequence edits is another target of this study. The introduction of activating mutations into the KIT gene in BEL-A2 cell line may help to reduce the high cost of erythroid cell culture, also allowing the eventual production of red blood cells, to be more economically attainable worldwide.
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