Event Title

Expression analysis through silencing of D930015E06Rik during erythroid terminal differentiation

Location

Edwards 107

Document Type

Oral/PowerPoint Presentation (10 minutes and 5 minute Q and A)

Description

Erythroid terminal differentiation (ETD) is the process whereby erythrocyte precursors develop into mature red blood cells (RBCs). Differences in gene expression are responsible for causing this maturation. Expression of D930015E06Rik or Late Erythroblast-3 (LEB-3) increases during ETD suggesting that this gene may play a critical role in the maturation process. The function of LEB-3 in ETD is as yet unknown, but we hypothesize that its impaired expression would lead to errors in ETD. In order to study the potential role of LEB-3 in ETD, silencing of the LEB-3 gene has been performed by transfection of a plasmid vector expressing shRNA specific for LEB-3 into Murine Erythroleukemia (MEL) cells. MEL cells mimic ETD in vitro upon induction with DMSO. The maturation of the silenced cells will be compared with the maturation of normal MEL cells and MEL cells transfected with non-specific shRNA. A difference in the maturation pattern in the silenced MEL cells would confirm a role of LEB-3 in ETD. Studies are currently underway to document changes in LEB-3 expression by qPCR and western blotting. A full length LEB-3 cDNA has also been introduced into MEL cells to obtain a system overexpressing the gene. These cells will act as a positive control for mRNA and protein expression and also be used to investigate the effect of overexpression of LEB-3 during ETD. Determination of the function of LEB-3 during ETD will help in understanding how RBCs are formed and may thus prove to be important in understanding certain hereditary anemias in humans.

Start Date

April 2014

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Apr 26th, 2:10 PM

Expression analysis through silencing of D930015E06Rik during erythroid terminal differentiation

Edwards 107

Erythroid terminal differentiation (ETD) is the process whereby erythrocyte precursors develop into mature red blood cells (RBCs). Differences in gene expression are responsible for causing this maturation. Expression of D930015E06Rik or Late Erythroblast-3 (LEB-3) increases during ETD suggesting that this gene may play a critical role in the maturation process. The function of LEB-3 in ETD is as yet unknown, but we hypothesize that its impaired expression would lead to errors in ETD. In order to study the potential role of LEB-3 in ETD, silencing of the LEB-3 gene has been performed by transfection of a plasmid vector expressing shRNA specific for LEB-3 into Murine Erythroleukemia (MEL) cells. MEL cells mimic ETD in vitro upon induction with DMSO. The maturation of the silenced cells will be compared with the maturation of normal MEL cells and MEL cells transfected with non-specific shRNA. A difference in the maturation pattern in the silenced MEL cells would confirm a role of LEB-3 in ETD. Studies are currently underway to document changes in LEB-3 expression by qPCR and western blotting. A full length LEB-3 cDNA has also been introduced into MEL cells to obtain a system overexpressing the gene. These cells will act as a positive control for mRNA and protein expression and also be used to investigate the effect of overexpression of LEB-3 during ETD. Determination of the function of LEB-3 during ETD will help in understanding how RBCs are formed and may thus prove to be important in understanding certain hereditary anemias in humans.