Affinity purification, expression analysis, and cell line generation of D930015E06Rik
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Anemia, or insufficient production of red blood cells (RBC), affects close to 3.5 million people in the U.S. Gene D930015E06Rik, also named late erythroblast 3 (LEB-3) is highly expressed during the later stages of erythroid terminal differentiation, which is the process whereby RBC are produced in the body. The purpose of this project to develop the tools to determine D930015E06Rik's function through: development of a knockdown assay of function in cells via siRNA to see what effect the loss of expression would have on ETD and the development of controls to access its location in erythroid progenitors through western blot and immunofluorescence analyses. Previous studies achieved cloning and expression of a portion of the coding sequence of D930015E06Rik cDNA as a thioredoxin fusion protein in E. coli. The recombinant partial D930015E06Rik (rLEB-3) fusion protein was cleaved from thioredoxin and reacted successfully with a goat anti-rabbit D930015E06Rik antibody that was generated from the rLEB-3 fusion protein. This antibody is currently being used in experiments to document expression of the wild type D930015E06Rik protein in Friend Virus Anemia cells, as well as in continuous murine erythroleukemia (MEL) cell line. MEL cells can be induced to undergo a limited form of ETD, but allow for generation of stable transfected cell lines. A full sequence synthetic clone of D930015E06Rik was expressed using the GeneSwitchTM system within MEL cells. Short hairpin RNA (shRNA) specific for D930015E06Rik were generated and will be transfected into the MEL cells to knock down expression of the wild type D930015E06Rik. Future studies will assess the knock down of D930015E06Rik during ETD using western blot analysis as well as quantitative polymerase chain reaction (qPCR). Effects of D930015E06Rik knock down on the ETD of MEL cells will be assessed in the hope of determining the function of D930015E06Rik in this process, which may, contribute to improvement of therapies for anemia.