Inhibition of apolipoprotein-L 11b (Apol11b) gene expression using small interference RNA technology during erythroid terminal differentiation
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Erythroblasts differentiate and lose their nucleus to become functional erythrocytes by a process called erythroid terminal differentiation. It involves a number of processes, including decrease in cell size and volume, condensation and inactivation of chromatin, degradation of organelles and proteins, membrane skeletal remodeling and extrusion of the nucleus. The glycoprotein hormone erythropoietin (EPO) is instrumental in stimulating erythroid terminal differentiation while preventing programmed cell death, known as apoptosis. Apol11b, a novel protein, has been found to increase during the late stages of red blood cell differentiation and formation. It is hypothesized Apol11b may be a lipid binding protein that plays a role in the alteration of membrane lipid composition. This hypothesis was tested by attempting to inhibit Apol11b protein production by using small interference RNA (siRNA) technology. This involved the insertion of small interference RNA into a cell model, Friend erythroleukemia virus (FVA cells) by electroporation. An antibody directed against the Apol11b gene product was used to analyze the inhibition of Apol11b by western blot analysis. Results indicated that Apol11b was not knocked down and further optimization of electroporation conditions was required. Successful inhibition of Apol11b would help us determine the role played by Apol11b during the later stages of differentiation and further elucidate the mechanisms involved in these processes.