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Genome-wide CRISPR screen to determine
the identity of the genes that mediate the functional consequences of Hepatic Leukemia Factor (Hlf) Masters Degree project in General Molecular Biology, 30 credits Oguzhan Ergün Supervisor: David Bryder Co-supervisor: Martin Wahlestedt The Section of Developmental Hematopoesis Division of Molecular Hematology Project runs: – In this project, I will try to determine the identity of the genes that are responsible for mediating the functional consequences of Hlf expression. Since Hlf is a DNA binding transcription factor (TF) these genes may be either direct or secondary targets of Hlf DNA-binding, or may alternatively be interaction partners. Background; Hlf encodes a DNA binding transcription TF belonging to a subfamily of basic leucine zipper (bZIP) TFs that contain proline acidic-rich (PAR) domain and bind DNA through homo- and heterodimerization with any of its family members including Dbp, Tef and Nfil3 (Hunger et al., Molecular and Cellular Biology, 1996). Hlf has been found to give rise to a form of a childhood B-cell leukemia when fused with the gene E2A through the translocation t(17;19) (Hunger et al., Mol. and Cell. Biology, 1996). However, no published study has identify the role of Hlf in normal physiological blood cell formation. Previous unpublished studies suggested that Hlf is an important candidate regulator in early cell fate decision To investigate this, Hlf inducible GMLPs will be isolated and transduced with a genome wide CRISPR knockout library (GECKO v2). After transduction cells will be cultured on OP9 stromal cells and allowed to differentiate towars B lymphocytes. Isolation of these B cells by FACS will followed by genomic DNA purification and PCR amplification. Finally, these amplicons will be subjected to deep sequencing and evaluated to find out which gene(s) were knocked out that mediate in this differentiation process. Significance: To identify the candidate genes regulated by Hlf and involve in early cell decision in hematopoiesis will may lead better understanding of transcriptional network of hematopoiesis. References 1. Bryder D, Rossi DJ, Weissman IL.Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. The American Society for Investigative Pathology. 2006;169(2): 2. Hunger SP, Brown R, Cleary, M L. DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF. Molecular and Cellular Biology .1994;14(9):
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