Expression analysis of late erythroblast-1 during erythroid terminal differentiation
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Erythroid terminal differentiation is the process during which immature precursor cells undergo differentiation and form mature reticulocytes. During this process, a number of changes occur in the cell, one of which is the alteration in the levels of lipid composition in the erythrocyte membrane. Apolipoprotein L 11b Apol11b, a novel protein, was hypothesized to play a role in this alteration. Further investigation was to be done by implementing RNA interference to knockdown the expression of the protein to assess the effect of the knockdown on the process of erythroid terminal differentiation. Two cell models were used for the purpose of this study: Murine erythroleukemia cells (MEL) and Friend virus anemia (FVA) cells. Murine erythroleukemia cells were initially used because they are a continuous cell model and express Apol11b upon induction with Dimethylsulfoxide. Initial attempts to achieve RNA silencing were performed by using shRNA plasmid constructs in E.coli obtained from Dr. Te-Chung Lee (originally from Thermoscientific ® ). DNA from these constructs was purified for transfection into MEL cells via electroporation, however, when sequencing was performed, the DNA from these constructs was not a 100% complementary to the Apol11b gene. The next method used was the shRNA plasmid constructs obtained from Santa Cruz Biotechnology and these constructs were heat shocked into E.coli and the DNA was purified for electroporating into MEL cells. However, the viability of the MEL cells was very low after addition of the selection antibiotic. Since linearizing the plasmid DNA would increase the success rate for electroporation, we contacted SCBT to provide us with the vector map. However, due to proprietary reasons, they failed to do so. This resulted in the use of a third method for the purpose of achieving RNA interference: the use of lentiviral particles (also ordered from Santa Cruz Biotechnology). We ordered lentiviral particles for both the Apol11b and the control to transduce MEL cells with the use of a cationic polymer called polybrene. Stable clones were collected one week from the day of introduction of the viral particles. RNA and protein was collected from these cells and the RNA was reverse transcribed to perform qPCR and the fold changes were recorded for four data sets. The statistical analysis of the three of four data sets showed that the silencing process was not significant. As a second part of the project, western blot analysis was done using protein fractions from the nucleus and the cytosol collected from FVA cells (days 0 and 4). The Apol11b protein expression increased progressively from day 0 to day 4 of erythroid terminal differentiation, which not only confirmed the location of the protein in the cytosolic fraction of the cell but also proved its increased expression during erythroid terminal differentiation.