Inhibition of late erythroblast-1 (LEB-1) gene expression using short interference RNA technology
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Erythroid Terminal Differentiation (ETD) is the process by which erythroblasts mature and form erythrocytes. In ETD, the precursor to a mature red blood cell (RBC) undergoes extensive physical and chemical changes. These changes are thought to be regulated by feedback mechanisms acting on enzymes and proteins within the cell. The more visible results of these changes to the cell include: a decrease in cell size and volume; degradation of cellular organelles, and the extrusion of the nucleus. Previous research has identified a novel protein, with no known function, named Late Erythroblast-1 (LEB-1) that increases in expression during the terminal stages of ETD but which is not expressed in mature RBC. We hypothesized that LEB-1 plays a role in the process of ETD. This hypothesis was to be tested through inhibiting LEB-1 protein production. The inhibition of LEB-1 protein production involved the insertion of a small interference RNA vector into Murine Erythroleukemia (MEL) cells used as an ETD model. Six short interfering RNA (siRNA) constructs were synthesized with four specific for LEB-1 and designed through software on the Ambion website. Two sequences were used for controls and included in the siRNA kit provided by Ambion. These included a positive control that would inhibit GAPDH expression in the MEL cells and a scrambled sequence negative control having no known homology to the murine genome. After transformation of E. coli with the expression vectors, plasmid DNA was isolated and sequenced. Sequencing revealed matches for two of the LEB-1 specific constructs, as well as the positive and negative controls. Electroporation was employed to introduce the 4 recombinant siRNA plasmids into MEL cells in order to assess whether LEB-1 protein production could be diminished. MEL cells carrying the positive and negative control siRNA plasmids were successfully transfected and selected. The plasmids containing the LEB-1 specific siRNA plasmids were not successfully transfected in the first electroporation attempt. The effect of the decrease in LEB-1 protein production was to be determined in later studies by Western blotting and correlating changes in LEB-1 expression with alterations in the ETD process.