A High Throughput Screening System for the Identification of Novel Genes in the Regulation of Mammalian Erythropoiesis

Abstract 2093

Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursors. This induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. A recent genome wide transcriptional profile study demonstrated that over 600 genes are up-regulated during erythropoiesis. Surprisingly, the roles that most of the identified genes play in erythroid cells are still unknown. Understanding the functions of these unknown genes in the erythroid cells is necessary to elucidate the pathogenesis of red cell disorders such as congenital dyserythropoietic anemias, fanconi anemia, aplastic anemia, megaloblastic anemia, as well as leukemia and myelodysplastic syndromes with leukemic or dysplastic erythroid features. The goal of our study is to identify novel genes involved in different stages of erythropoiesis. To achieve this goal, we developed a high-throughput flow cytometry based assay that simultaneously detects erythroid cell differentiation and enucleation. In this assay, mouse fetal liver erythroblasts were purified and infected with lentiviruses containing a mammalian shRNA knockdown library obtained from the Broad Institute. The infected cells were cultured in a 96-well plate. Over the following two days the unaffected cells fully differentiate with approximately 60% of the cells enucleated. However, those cells in which shRNAs have knocked down genes critical for erythropoiesis are expected to show alterations in differentiation and/or enucleation. The system was validated using lentiviruses expressing shRNAs against Gata1 and mDia2, known proteins that are involved in the early and late stages of erythropoiesis, which showed inhibitions of differentiation and enucleation, respectively. We have pre-screened the most up-regulated 100 genes that play unknown functions in the erythroid cells. Future studies will be focused on the identified novel genes on their functions in erythroid lineage commitment, Epo mediated signaling pathways, hemoglobin enrichment, chromatin condensation, and enucleation.