GENETIC DISEASE STRATEGY TO DEVELOP ANIMAL MODELS

开发动物模型的遗传疾病策略

• 批准号: 6116733
• 负责人: JEFFREY J WINE
• 金额: $ 4.41万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6116733
• 关键词: animal tissue; biotechnology; congenital disorders; inborn metabolism disorder; respiratory system

Significance Discovery of natural primate models of human recessive genetic diseases by the novel methods proposed herein will provide a new approach to the analysis and treatment of disease for which mouse models are inadequate. Objectives (1) To analyze the DNA of certain monkey genes to test the hypothesis that mutation frequencies in these regions are similar to frequencies observed in human populations, and (2) to breed the healthy heterozygous carriers to obtain homozygous disease models. Results Small amounts of blood are obtained during the routine, physical examinations of animals and genomic DNA is purified from the samples and analyzed using single stranded conformation polymorphism (SSCP) analysis. In this analysis, a fragment of DNA is amplified via polymerase chain reaction (PCR) in the presence of radiolabeled nucleotides, denatured, and run on a non-denaturing gel. When samples from many individuals are run side by side, all individuals having identical DNA sequences display identical patterns. Any departure from this pattern, including single base pair differences, can be detected with close to 100% fidelity. When such differences are detected, the altered DNA region from a representative animal is then sequenced. We have purified DNA from 330 rhesus and 12 cynomolgous monkeys housed at the CRPRC and have analyzed 25 exons for sequence variations. Numerous polymorphisms and species-specific differences have been detected, as well as several candidate missense mutations. The missense mutations were tested in a physiological assay that indicated reduced function. We have begun a program to breed carriers of the missense DNA changes. To date 7 animals are incorporated into the program, including one infant born in captivity as an offspring of a G239E carrier male and an I1269M carrier female. Future Directions Develop more efficient screening methods to allow parallel screening of many genes in larger populations of primates. KEY WORDS cystic fibrosis FUNDING NIH Grant DK51776

人类自然灵长类模型的意义发现 通过本文提出的新方法获得的隐性遗传病 为分析和治疗疾病提供了一种新的途径 哪些小鼠模型是不完善的。目标(1)分析DNA 某些猴子基因来验证这样的假设 这些区域的频率与在 人类群体；(2)培育健康的杂合子携带者 以获得纯合子疾病模型。结果少量的血液 是在例行的动物体检和 从样品中提纯基因组DNA，并用单个 链构象多态性(SSCP)分析。在这一分析中， 通过聚合酶链式反应(PCR)扩增出一段DNA 放射性标记核苷酸的存在，变性，并在一个 非变性凝胶。当来自多个个体的样本被放在一边时 并排显示具有相同DNA序列的所有个体 一模一样的图案。任何偏离这一模式的行为，包括单一 碱基对差异，可以接近100%的保真度被检测到。 当检测到这种差异时，来自 然后对具有代表性的动物进行测序。我们已经提纯了DNA 330只恒河猴和12只食蟹猴被安置在CRPRC，并有 分析了25个外显子的序列变异。大量的多态和 已经检测到特定物种的差异，以及几个 候选错义突变。错义突变在一项 表明机能减退的生理学测试。我们已经开始了一场 培育错义DNA变化携带者的计划。到目前为止7 动物被纳入该计划，包括一名出生的婴儿 作为G239E携带者和I1269M携带者的后代被圈养 航母。未来方向发展更有效的筛查 允许在更大范围内并行筛选多个基因的方法 灵长类动物的种群。关键词囊性纤维化基金美国国立卫生研究院赠款 DK51776