Numerous cellular studies designed to elucidate the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR) and Cl ion transport employ a full-length cDNA that produces a functional protein product. Because these investigations involve overexpression of CFTR, they cannot evaluate the role that CFTR levels play in the expression of a given ion transport phenotype nor can they assess the effect of somatic genetic factors on expression of the CF phenotype. The project outlined here will use homologous recombination as an alternative to gene complementation with cDNA and will develop cell lines that have specific CF genotypes. This approach to understanding CFTR function has several advantages: 1) it places exogenous CFTR sequences under the regulation of the endogenous CFTR promoter and ensures that CFTR is expressed at appropriate levels in a given cell type, 2) it facilitates analysis of different CFTR mutations in a constant genetic background, i.e., one mutation will be replaced by another in the same cell line, and 3) it uses a human cell system that normally expresses CFTR. Transformed normal and cystic fibrosis epithelial cells developed in this laboratory will be used in these studies. Initial complementation studies will use a CF cell line, homozygous for the delta-F508 mutation. Homologous recombination vectors containing genomic CFTR sequences will be introduced into the cells by electroporation. The incoming genomic CFTR sequences will cover a region of CFTR that includes exon 10 and the flanking 5' and 3' intron sequences. Two strategies for targeted gene replacement will be used. One strategy will employ replacement vectors. The replacement vector, will be constructed such that the neo(r) gene is contained within flanking intron sequences. The HSV-tk gene will be adjacent to the region of homology and will therefore be eliminated during homologous recombination. Homologous recombinants will be selected by a positive/negative selection (PNS) scheme that enriches for homologous recombinants. PNS relies on positive selection for G418 resistance (neo(r)) and negative selection for the presence of the herpes simplex virus thymidine kinase (HSV-tk) gene. The other approach will use insertional vectors and rely on intrachromosomal recombination to eliminate the selection markers and duplicate genomic sequences. CF cell lines with a specific CF genotype will be generated by homologous replacement of one CF allele with another. Homologous recombinants will be characterized for their Cl ion transport properties by efflux and patch clamp analyses measuring 36CI efflux and whole-cell and single channel Cl currents, respectively. In addition, recombinant cells will be assayed for expression of CFTR mRNA and protein to determine the role that a specific CFTR mutation has on the level of CFTR expression and the associated phenotype.
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