Inhibition of late erythroblast-3 (LEB-3) gene expression using small interference RNA technology
Dean, Rebecca E.
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Erythroid Terminal Differentiation (ETD) is the process by which red blood cell precursors (erythroblasts) form mature red blood cells (erythrocytes). Precursor cells undergo extensive physical and chemical changes during this process. These include: decrease in cell size and volume; degradation of cellular organelles, and the extrusion of the nucleus. In order for RBC differentiation to begin, the glycoprotein hormone erythropoietin (EPO) must be present. EPO allows RBC precursors to undergo ETD, by preventing the cells from undergoing programmed cell death (apoptosis). A novel protein named Late Erythroblast-3 (LEB-3) is strongly expressed during the terminal stages of ETD, suggesting that LEB-3 plays a role in ETD. LEB-3 protein production was inhibited through the insertion of small interference RNA (siRNA) into Murine Erythroleukemia (MEL) cells, to determine what effect the loss of LEB-3 had on ETD. Preliminary work in the lab resulted in the generation of several MEL cell lines containing plasmids that are capable of producing siRNAs that could inhibit LEB-3 protein production. An antibody directed against LEB-3 was generated to assess the level of inhibition LEB-3 protein in the MEL cell lines by Western Blotting. This will allow us to determine which siRNAs will have the greatest effect upon disrupting LEB-3 protein production, and if there are any associated alterations in ETD process.