GENETIC DISEASE STRATEGY TO DEVELOP ANIMAL MODELS

人类自然灵长类动物模型的发现 通过本文提出的新方法， 为疾病的分析和治疗提供了一种新的方法， 哪种小鼠模型是不够的。 目的（1）分析DNA 来验证突变 在这些地区的频率是类似的频率观察到， 人类群体，和（2）培育健康的杂合携带者 以获得纯合疾病模型。 结果少量血液 在动物的常规体格检查中获得， 从样品中纯化基因组DNA，并使用单克隆抗体进行分析。 链构象多态性（SSCP）分析。 在本分析中， 通过聚合酶链式反应（PCR）扩增DNA片段， 放射性标记的核苷酸的存在，变性，并运行在一个 非变性凝胶。 当来自许多个体的样品同时运行时， 与此同时，所有具有相同DNA序列的个体 相同的模式。 任何偏离这种模式的行为，包括单身 碱基对差异，可以检测到接近100%的保真度。 当检测到这种差异时，来自一个人的改变的DNA区域 然后对代表性动物进行测序。 我们已经纯化了 330只恒河猴和12只食蟹猴饲养在CRPRC， 分析了25个外显子的序列变异 许多多态性和 已经发现了物种特异性差异，以及一些 候选错义突变。 错义突变在一个 生理分析表明功能降低。 我们已经开始了 培育错义DNA携带者的计划。 迄今为止7 动物被纳入该计划，包括一个婴儿出生 作为G239E携带者雄性和I1269M携带者的后代被圈养 女性携带者 发展更有效的筛选 允许在较大的细胞中平行筛选许多基因的方法 灵长类动物的种群。 关键词囊性纤维化 DK51776

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