Expression of D930015E06Rik and components of the autophagy system during terminal erythroid differentiation
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This project focuses on the expression of the D930015E06Rik gene in erythrocytes. A secondary aim is to obtain pilot data regarding various other genes that are up/down-regulated during this process of autophagy in erythroid terminal differentiation. Central to this project was the previous work involving the development of antibodies to D930015E06Rik gene. To validate this antibody it was very important to develop a positive control for the characterization of D930015E06Rik gene. For the development of a positive control, primers were designed to amplify the cDNA of the D930015E06Rik gene sequence and cDNA was amplified by RT-PCR. Amplified cDNA was cloned into a TOPO vector and then this clone was transformed into E.Coli cells. LB agar plates were cultured and the transformants grown on those plates. Plasmids were isolated from transformants and restriction digestion was carried out to make sure that the gene was incorporated correctly. DNA samples were then sent for sequencing which matched perfectly with the amplified cDNA. To show that induction was also possible and the amount of protein expressed kept on increasing as time progressed, an induction experiment was carried out using IPTG as the inducing agent. Bands were analyzed on a gel and the molecular weights calculated. A western blot was also run to prove that the antibodies indeed recognized this induced protein and prove that the peptide sequence was correctly cloned into the plasmid. Finally PCR arrays from SABiosciences were run to find out genes up/down regulated during the autophagy process in ETD. The data from the qPCR machine was uploaded onto the SABiosciences website and genes which were up/down regulated during the autophagy process were noted. This was done because previous studies had indicated that the mitochondria, nucleus and other organelles were extruded form the cell by the process of autophagy. The future work would include the affinity purification of the antibody to produce the positive control and then run it on western blots to characterize the molecular weight of the D930015E06Rik gene and find out its exact function. Similar experimental lines can be done for the various genes involved in autophagy.