The long term objective of our research is to identify the basic defect in Cystic Fibrosis (CF). A necessary step to accomplish this goal is to be able to identify the CF gene from among candidate genes isolated by virtue of their location in the region of the CF locus.
The specific aims of this application are to establish transformed epithelial cell lines derived from patients with Cystic Fibrosis (CF) and control donors and to develop methods to assay candidate CF genes. We will therefore first transform and characterize primary epithelial cell from affected and unaffected individuals. We will then use these cells to establish the best method for the introduction of heterologous DNA and develop a microelectrode puncture method using fused cells to detect a CF- specific cellular phenotype. Primary cultures derived from a total of 12 sweat glands and nasal polyps will be infected with an adeno- SV40 recombinant virus and the transformed cells isolated for further characterization. The long term growth of the cells will be determined by continuous subculture. The epithelial origin of the cells will be confirmed by electron microscopy and immunocytochemistry. The presence of the transforming SV40 gene will be detected by immunocytochemistry and analysis of genomic DNA. The electrophysiological properties of the transformed cells will be studied with the use of patch clamping techniques to confirm the expression of Cl- channels and their aberrant regulation in CF cells. We will develop a microelectrode technique for routine screening of the electrical properties of the cells. Microelectrode puncture measurements will be done on homokaryons consisting of 10-100 fused cells produced by treatment of the cultures with polyethylene glycol. This latter technique should be easier to use in laboratories not set up for sophisticated electrophysiological measurements. The best technique for the transfer of DNA into the cells will be assessed by studying the expression of the bacterial chloramphenicol acetyl transferase and neo genes after DNA-mediated gene transfer with vectors carrying these genes under the control of mammalian promoters. These cell lines and technique will be used for testing candidate CF genes as they become available. In addition, the cell lines and the assay system will be a valuable resource for experiments to be conducted after the CF gene is identified. They will be used to analyze the nature of regulation of the CF gene, for functional studies of the CF gene product and for the testing of therapies in which the CF cellular phenotype is modulated.
| Tsui, L C; Buchwald, M (1991) Biochemical and molecular genetics of cystic fibrosis. Adv Hum Genet 20:153-266, 311-2 |
| Buchanan, J A; Yeger, H; Tabcharani, J A et al. (1990) Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals. J Cell Sci 95 ( Pt 1):109-23 |
