The single human erythropoietin gene, located on chromosome 7, consists of 5 exons spanning 3 kb of genomic DNA. In human, rodents and sheep, erythropoietin is produced by the fetal/neonatal liver, followed by a gradual transition to primary production by the adult kidney. 1.6 and 2.0 kb RNA species have been identified in preparations from human liver. Comparison of the human and murine erythropoietin gene sequences has revealed a striking degree of homology within amino-acid coding sequences. In addition, specific 5' - and 3' -flanking sequences as well as sequences within the first intron of the gene are highly conserved, suggesting that these sequences may have functional significance. The purpose of this study is to utilize the newly-developed technique of pronuclear microinjection to analyze the DNA sequences involved in the tissue and developmental-stage specific regulation of the human erythropoietin gene. We will first test the hypothesis that transgenic mice harboring a 4 kb human genomic DNA fragment encompassing the erythropoietin gene will coordinately express the endogenous murine erythropoietin gene and the transgene. Important parameters that will be studied include the ability of the transgene to undergo normal regulation as measured by developmental-stage and tissue-specific expression and response to inductive stimuli such as hypoxia. The second stage of the study will involved the identification of the DNA sequences involved in the regulation of erythropoietin gene expression. Deletion mutants lacking specific sequences which are conserved between the murine and human erythropoietin genes will be constructed and introduced into transgenic mice. Expression of these altered transgenes will be compared to that of the intact transgene in terms of tissue and developmental specificity. The results of these studies should have significance with respect to our general understanding of the control of gene expression, as well as more specific questions regarding the hormonal regulation of hematopoiesis, during development.
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