CHARACTERIZATION OF THE MURINE HOMOLOG FOR THE HUMAN P47-PHOX GENE

这个非临床的IIDEA项目的目的是识别、克隆和 鉴定与人类P47-Phox基因相对应的小鼠基因 常染色体隐性遗传性慢性吞噬细胞蛋白缺陷的编码 肉芽肿性疾病。没有人类受试者参与其中。一种抗人的抗体 重组p47-Phox在小鼠体内检测到一个大小几乎相同的蛋白质 骨髓。一系列哺乳动物、禽类和酵母DNA的DNA分析 发现了与人类基因同源的DNA片段。灵长类动物中 与小鼠的DNA相似，但不那么相似。从两只小鼠体内提取RNA的研究 细胞系1C21和WEHI-3检测到小鼠的mRNA转录 P47-Phox基因大小约为2.5-3.0kb。为了克隆这个基因，一个 从WEHI-3细胞中分离的mRNA构建的c DNA文库用 放射性标记的人p47-Phox基因探针。阳性克隆是 提纯和鉴定。5个克隆被鉴定为2.6-2.8 kb 在尺寸上。对这些克隆进行DNA序列分析。比较 小鼠和人类的DNA序列显示出80%-85%的一致性 小鼠基因和人类基因在编码基因区域 蛋白。在大多数情况下，碱基差异出现在第三个或 三联体密码子的“摆动”位置，不产生氨基酸 蛋白质的变化。在未来一年里，利用基因靶向 技术，小鼠的基因将被同源基因原位突变 胚胎干细胞(ES)系中的重组产生多能性 缺乏小鼠胞浆蛋白的细胞系。这些ES细胞可以是 注射到小鼠囊胚中，产生携带异常病毒的小鼠 吉恩。这些动物将提供一个模型系统来研究 炎症反应和从基因上纠正他们的缺陷。这 将提供有关纠正人类基因的潜在方法的信息 未来的缺陷。

The purpose of this non-clinical, IIDEA project is to identify, clone, and characterize the murine gene corresponding to the human p47-phox gene which codes for the phagocyte protein deficient in autosomal recessive Chronic Granulomatous Disease. No human subjects are involved. An antibody to human recombinant p47-phox detects a protein of nearly identical size in murine bone marrow. DNA analysis of a series of mammalian, avian,and yeast DNA's revealed DNA fragments homologous to the human gene. Primates were most similar and murine DNA less so. Studies of RNA isolated from two murine cell lines 1C21 and WEHI-3 detected mRNA transcripts for the murine p47-phox gene of about 2.5-3.0 kilobases in size. To clone this gene, a cDNA library made from mRNA isolated from WEHI-3 cells was screened using a radioactively labelled human p47-phox cDNA probe. Positive clones were purified and characterized. Five clones were identified as being 2.6-2.8 kb in size. These clones were subjected to DNA sequence analysis. Comparison of the murine and human DNA sequence has revealed 80-85% identity between the murine gene and the human gene in the region of the gene coding for protein. In the majority of cases, base differences occur in the third or "wobble" position of the triplet codon and do not result in amino acid changes in the protein. In the coming year, utilizing gene targeting techniques, the murine gene will be mutated in situ by homologous recombination in an embryonal stem (ES) cell line creating a pluripotent cell line deficient in the murine cytosolic protein. These ES cells can be introduced into murine blastocysts to generate mice carrying an abnormal gene. These animals will provide a model system for studying the inflammatory response and for genetically correcting their defect. This will provide information on potential ways to correct the human genetic defect in the future.