Gamma globin silencer gene(s) will be identified in an animal model of sickle cell disease (SCD) after global mutagenesis. N-ethyI-N-nitrosourea (ENU) will be used to induce point mutations that will inactivate or alter random sets of genes throughout the mouse genome in individual embryonic stem (ES) cells. Mice that are cloned from these cells will be screened for dominant and recessive mutations that affect gamma globin gene silencing by quantifying the persistent expression and synthesis of fetal hemoglobin in the founder animals and their progeny. This phenotype driven approach will utilize a knockout-transgenic mouse model of SCD that reproduces most if not alt of the pathology of the disorder (Science 278: 873-876). The model was created by targeted deletion of the adult mouse alpha and beta globin genes followed by introduction of human alpha, gamma, and beta sickle globin transgenes into the germline. During fetal and adult life these animals synthesize only human hemoglobin in their red blood cells. Similar to man, these SCD mice switch from human fetal hemoglobin, HbF, to adult sickle hemoglobin, HbS, at the time of birth. SCD mouse ES cell lines will be established from developing blastocysts isolated from sickle cell females that were mated with sickle males. Sickle ES cells will be treated with ENU and thousands of individual subclones established. Hundreds of SCD founder mice will be produced annually from these randomly mutated ES cell subclones by tetraploid embryo complementation. Alterations of heterocelluiar gamma globin chain levels in circulating erythrocytes will be assessed in founder animals and their offspring to discover potential cell lines containing gamma globin silencer mutations. Microsatellite linkage analysis of mutant offspring outcrossed to congenic SCD mice and direct sequence comparison to the routine genome will allow the positional cloning of gamma globin silencer genes. Finally, putative silencing factors will be positively confirmed by replicating the exact germline modification discovered during the ENU screen into SCD and beta thalassemic ES cells, followed by direct examination of the phenotype in mice generated from the modified cells by cloning. Successful completion of these studies will define the gene(s) responsible for gamma globin silencing. By experimental design, the in vivo therapeutic benefits associated with increasing HbF levels due to modification of these gone(s) on the pathophysioiogy of sickle cell anemia and Cootey's anemia will be tested directly in animal models of these disorders.
