EXPRESSION OF HUMAN ERYTHROPOIETIN GENE

位于7号染色体上的单个人类促红细胞生成素基因， 由跨越3 kb基因组DNA的5个外显子组成。 在人类中， 啮齿动物和绵羊，红细胞生成素是由 胎儿/新生儿肝脏，然后逐渐过渡到原发性 由成人肾脏产生。 1.6和2.0 kb RNA物种具有 在人类肝脏的制剂中被发现。 比较 人类和鼠促红细胞生成素基因序列已经揭示了 氨基酸编码中惊人的同源性 序列的 此外，特异性的5' -和3' -侧翼序列作为 以及基因的第一内含子内的序列高度 保守，这表明这些序列可能具有功能性 意义 本研究的目的是利用新开发的 原核显微注射DNA分析技术 参与组织和发育阶段特异性 人促红细胞生成素基因的调控。 我们将首先测试 携带4kb人类基因的转基因小鼠 包含促红细胞生成素基因的基因组DNA片段将 协同表达内源性鼠促红细胞生成素基因 和转基因。 将研究的重要参数 包括转基因进行正常调节的能力 通过发育阶段和组织特异性 表达和对诱导刺激如缺氧的反应。 研究的第二阶段将涉及确定 参与促红细胞生成素调节的DNA序列 基因表达。 缺乏特定序列的缺失突变体 在鼠和人之间是保守的 将构建促红细胞生成素基因并将其导入 转基因小鼠 这些改变的转基因的表达将是 与完整的转基因相比， 发育特异性 这些研究的结果应该 对我们对人类的普遍理解有重要意义 基因表达的控制，以及更具体的问题 关于造血的激素调节， 发展

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.