Silencing and overexpression of D930015E06Rik during erythroid terminal differentiation
Odak, Ashlesha P.
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Background: Erythroid terminal differentiation (ETD) is the process by which erythrocyte precursors develop into mature red blood cells (RBCs). Differences in gene expression are responsible for bringing about this maturation. D930015E06Rik or Late Erythroblast-3 ( LEB-3 ) is a gene found on the mouse chromosome 3. This gene was shown to have a significantly higher expression in maturing erythroblasts of Friend Virus Anemia (FVA) cells as the maturation to the reticulocyte stage progressed. LEB-3 shares 77% homology to a novel gene in humans with a high level of nucleotide conservation in other animal species. The function of the protein produced from this gene and its location within the cell are as yet unknown. However, upregulation of this gene during ETD suggests that this gene may play a role in the maturation process of erythrocytes. Methods: Several experimental approaches were undertaken in order to study the effects of LEB-3 silencing or LEB-3 overexpression in the process of ETD in Murine Erythroleukemia (MEL) cells. MEL cells mimic ETD in vitro upon induction with DMSO. The first approach was to construct a cell model to silence the expression of LEB-3 by introduction of shRNA specific for the LEB-3 through a plasmid vector into MEL cells. The maturation of the LEB-3 silenced MEL cells was compared with the maturation of the normal MEL cells through microscopy to observe morphological differences. Quantitative Polymerase Chain Reaction (qPCR) with the isolated RNA was used to quantitate the difference in gene expression between the two cells. A second experimental approach was to transfect an inducible LEB-3 expression plasmid into MEL cells through electroporation. A system overexpressing LEB-3 would help to study the effects of overexpression of LEB-3 in undifferentiated MEL cells and MEL cells undergoing differentiation. These cells would also be a useful tool as a positive control for developing immunologic assays to localize LEB-3 protein. Results: qPCR studies showed that both LEB-3 and β globin (a marker of differentiation) displayed higher expression as differentiation progressed in MEL cells as compared to the silenced MEL cells. Microscopy of both cells showed that there was a difference in the morphological characteristics between the MEL cells and silenced MEL cells in the uninduced as well as induced state. MEL cells transfected with the overexpression vector were successfully selected with the selective antibiotic. An SDS-PAGE gel was run with the induced proteins; however, no particular band could be observed at the expected size. Conclusion/Discussion: As β globin production is essential to hemoglobin production, the decrease in the expression of β globin with a simultaneous decrease in LEB-3 expression in silenced cells suggests that LEB-3 may be essential in the process of ETD. Silencing of LEB-3 might also play a role in general morphological characteristics as well as differentiation in MEL cells. Stable cell lines have been established for the overexpression of LEB-3 , however, results obtained by SDS PAGE need to be confirmed by western blotting and qPCR. Immunolocalization and western blotting of LEB-3 are currently underway to localize the recombinant LEB-3 protein in transfected MEL cells.