Production of Polyclonal anti- D930015E06Rik Antibody and Focused PCR Array Studies of Autophagy Pathway Related Genes During Erythroid Terminal Differentiation
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Erythroid terminal differentiation (ETD) is a process by which immature precursor cells become mature erythrocytes. The proerythroblast goes through various stages of cell differentiation, losing its organelles and nucleus, and decreasing in size, to form a mature erythrocyte. Previous studies in the lab identified 3 differentially expressed unknown genes during the process of ETD. D930015E06Rik (Late Erythroblast - 3) is one of those genes. D930015E06Rik is also expressed in the region of the testis where secondary spermatocytes and early spermatids are found, thus implying a role in spermatogenesis as well as ETD. Previous work involved the cloning of a partial coding region of mouse D930015E06Rik cDNA into pET 102/D-TOPO ® expression plasmid. One of the current research goals was to express this partial sequence and produce a polyclonal antibody against it. The recombinant D930015E06Rik partial protein was produced in E. coli and purified by taking advantage of the Ni + binding properties of the 6X histidine tag attached to the target protein. Two rabbits were immunized for the production of polyclonal anti-D930015E06Rik antibody, which was then analyzed for its D930015E06Rik partial protein specificity by Western blot. Though results are preliminary at this time, the serum from at least one rabbit was found to identify the positive control (purified protein) successfully. Future studies could involve D930015E06Rik protein cellular localization and gene knockdown effects, which would give significant evidence for the existence of the currently hypothetical D930015E06Rik gene. A second aim of this project was to investigate the autophagy pathway related gene expression during ETD, as these genes are likely to play an important role in the extrusion of cell nucleus and degradation of cellular organelles that occurs during the process. To understand the transcriptional (mRNA-level) effects, total RNA was obtained from mouse spleen FVA cells undergoing ETD, which was then used in autophagy-focused PCR array experiments. Out of the 84 autophagy pathway related genes, 15 were up-regulated and 3 were down-regulated, both groups having p-value<0.1. Future work will involve studies to understand the function and cellular localization of these genes with respect to ETD.