Inhibition of Apolipoprotein-L 11b ( Apol11b ) Gene Expression Using Small Interference RNA Technology During Erythroid Terminal Differentiation
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Erythroid Terminal Differentiation (ETD) is the process by which immature precursor cells differentiate and become mature erythrocytes. The process includes the synthesis and accumulation of hemoglobin, chromatin condensation, and extrusion of the nucleus. Additional alteration in lipid composition of erythrocyte membrane during this process may be influenced by a novel protein, Apol11b. The expression of this protein was to be disrupted using small interference RNA (siRNA) in this study. Murine Erythroleukemia (MEL) cells normally express Apol11b upon induction with dimethylsulfoxide and thus were used as a model for determining the level of silencing of the Apol11b gene using siRNA. Initial experiments were performed to optimize electroporation conditions using a green fluorescent protein (GFP) expressing plasmid, with GFP expression being used to assay success of electroporation. Inconsistent results from these experiments resulted in the application of chemical transfection and then finally lentiviral transduction methodologies. Replication-incompetent lentiviral particles with 3 different DNA constructs (GFP cDNA, Control shRNA and Apol11b shRNA ) were successfully used to transduce the MEL cells and stable cell lines were generated. Stable clones containing GFP confirmed successful transduction through visible fluorescence under microscope. Western blotting with an antibody previously generated against Apol11b protein and quantitative Polymerase Chain Reaction (qPCR) were used to assess Apol11b knockdown. Though the western blotting results could not be interpreted, statistical analysis of qPCR experiments demonstrated effective silencing of the Apol11b gene in the MEL cells transduced with the Apol11b shRNA vs. nontransduced MEL cells.