Sickle cell anemia is caused by a single base-pair substitution in codon 6 of the human beta-globin gene resulting in a mutant hemoglobin molecule. Hemoglobin S (HbS) polymerizes when deoxygenated and causes erythrocytes to deform and take on a characteristic """"""""sickled"""""""" shape. An animal model for sickle cell disease wig not only allow for the analysis of in vivo erythrocyte sickling and for more detailed study of the pathophysiology of the disease, but will permit the development of potential therapeutic modalities to treat the disease and prevent the accompanying complications. Previous attempts to develop such a transgenic model have failed to achieve an anemic animal with morphologic erythrocyte changes, accompanying histopathological changes and tissue damage as evidence of phenotypic expression of the disease. Using two novel approaches, we may be able to upregulate the expression of the exogenous alpha- and beta-like globin genes in a coordinated fashion and increase the propensity toward intracellular polmerization resulting in a pathologic transgenic phenotype. The variant sickle cell gene that we are using is hemoglobin S-Antilles, a2b2(6glu->val,23val->ile). This double mutant variant polymerizes more readily than sickle hemoglobin and results in disease manifestations in human heterozygous carriers of the disease. Constructs have been made using the human beta S-Antilies and a2 genes tandemly linked downstream of either two or four erythroid specific DNase I super-hypersensitive (HS) sites. These constructs have been injected into fertilized mouse embryos. Using isoelectric focusing, blood samples have been analyzed for the presence of human hemoglobin. Screening has demonstrated that three founder and two F2 mice are expressing human hemoglobin at levels of 15-20% of endogenous mouse hemoglobin. Quantitation of human and mouse globin chains has been determined by denaturing Triton-urea polyacrylamide gel electrophoresis. Transcription analysis is being performed using RNase protection assays and gene copy number has been determined by Southern analysis. Data on erythrocyte functional properties will include morphological analysis, oxygen affinity curves, hematological values and rheological studies. Further cross breeding of these animals should result in a line of transgenic animals manifesting the sickle cell phenotype.
