GENETIC DISEASE STRATEGY TO DEVELOP ANIMAL MODELS

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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