Until recently there have been no known animal models for cystic fibrosis. With isolation of the mouse cystic fibrosis gene it became possible to develop mouse models of cystic fibrosis. Three mouse models have been reported and were produced by disrupting the CFTR gene through homologous recombination. While two of the animals are gene """"""""knockouts"""""""" with distinctive gastrointestinal manifestations of CF, the other animal has duplicate homologous sequences inserted adjacent to one another and has milder CF disease symptoms. The variation between the two types of animals indicates that CF phenotype will vary depending on the CFTR mutation. At present there are, however, no animals with specific CFTR mutations analogous to those in humans. Because the animals described above do not represent naturally occurring CFTR mutations, there will be limitations on what can be learned from them regarding the relationship between a given CF pathology and a defined CFTR mutation. We propose to use a novel gene targeting protocol to develop CF mice with distinct CFTR,exon 10 mutations. The small fragment homologous recombination (SFHR) strategy will introduce small fragments (392 bp) of homologous DNA in mouse 129 embryonic stem (ES) cells. The fragments will contain specific exon 10 mutations as well as new restriction enzyme cleavage sites not found in wild type (wt) exon 10. Homologous recombination will be assessed by polymerase chain reaction (PCR) amplification of the genomic locus spanning the homologous sequences (435 bp) and subsequent restriction enzyme digestion of the PCR products. Enriched populations of recombinant CF heterozygote ES cells will be obtained after PCR screening isolated ES cell colonies plated at clonal density. Clones identified as predominantly CF heterozygotes will be expanded for genomic DNA analysis and measurement of the wild-type:CF heterozygte ratio. Colonies that have a greater than 70% proportion of CF heterozygote cells will be used for blastocyst injection. Chimeric animals will be screened by PCR and then assessed for germline transmission. Heterozygote mice will be bred and CF homozygote animals will be phenotypically and genotypically characterized. PCR amplification and genomic Southern hybridization will be employed to verify the mutation status of the animals. The phenotypic status of the CF mice will be determined by morphological assessment of the disease pathology is various organs (e.g., intestinal obstruction, airway' secretion) and the Cl ion transport properties of the epithelium. Organs will be analyzed by light electron microscopy and isolated epithelial cells will be assayed for CAMP-dependent efflux of 36Cl.
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