EXPRESSION OF HUMAN ERYTHROPOIETIN GENE

人类单个促红细胞生成素基因，位于7号染色体上， 由5个外显子组成，跨越3kb的基因组DNA。在人类身上， 啮齿动物和绵羊，促红细胞生成素是由 胎儿/新生儿肝脏，随后逐渐过渡到初级肝脏 由成人肾脏产生。1.6和2.0 kb的RNA物种有 已在人肝制剂中发现。比较 人类和小鼠促红细胞生成素基因序列已经揭示 氨基酸编码中惊人的同源性 序列。此外，特定的5‘和3’侧翼序列如 以及基因第一内含子内的序列高度一致 保守，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 原核显微注射技术分析DNA 参与组织和发育阶段特异性的序列 人促红细胞生成素基因的调控。我们将首先测试 假设携带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 内源性小鼠促红细胞生成素基因的协同表达 还有转基因。将研究的重要参数 包括转基因经历正常调控的能力 通过发育阶段和组织特异性来衡量 对低氧等诱导性刺激的表达和反应。 研究的第二阶段将涉及确定 促红细胞生成素调控的DNA序列 基因表达。缺乏特定序列的缺失突变体 它们在小鼠和人类之间是保守的 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 与完整转基因相比，在组织和 发育专一性。这些研究的结果应该是 对我们的一般理解具有重要意义 基因表达的控制，以及更具体的问题 关于造血的激素调节，在 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.