Dominant and Pharmacologically Sensitized ENU Mutagenesis Screens Uncover Novel Regulators of Hematopoiesis and Model Hematopoietic Disease.

To generate new mouse models of human hematopoietic disease and increase our knowledge of the genetic networks that control hematopoiesis, we are performing dominant (generation 1 or G1) and pharmacologically-sensitized forward ethylnitrososurea (ENU) mutagenesis screens. Mice are phenotyped by saphenous vein peripheral blood analysis using an automated hematological analyzer. ENU is ideally suited to generating models of human disease and annotating gene function because the spectrum of mutations (point mutations generating leading to subtle amino acid substitutions, splicing errors, or premature termination) are similar to those often found in human disease. Furthermore, null mutations often do not represent the full extent of a gene’s function, requiring multiple alleles to fully define gene function. While dominant mutations can unequivocally cause some human diseases, often mutations in multiple genes interact and contribute to disease progression. Thus, we have developed sensitized screens that induce transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea) and analyzing the recovery in peripheral blood levels of red blood cells, white blood cells and platelets. This strategy enables identification of hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. The induced cytopenia recovery assay is also being used as a secondary phenotyping assay for some of our G1 dominant mutants. The combined dominant and sensitized screens have yielded 14 heritable dominant mutants to date plus four additional mutants in hereditary testing. The array of mutations that we are analyzing are models for the following diseases: polycythemia, thrombocythemia, leukocytosis, anemia, and thrombocytopenia. I will discuss the progress of the mutagenesis screen and several ENU mutants, including a novel mutation in the protein tyrosine kinase Jak2, leading to thrombocythemia. This point mutation in the protein kinase domain will help us to dissect the recently discovered role of Jak2 in Myeloproliferative Diseases including Essential Thrombocythemia.