Gene targeting of CFTR DNA in CF epithelial cells

K. Kunzelmann, J. Y. Legendre, D. L. Knoell, L. C. Escobar, Z. Xu, D. C. Gruenert

Research output: Contribution to journalArticlepeer-review

92 Scopus citations


A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wit homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell parch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.

Original languageEnglish (US)
Pages (from-to)859-867
Number of pages9
JournalGene Therapy
Issue number10
StatePublished - 1996
Externally publishedYes


  • Gene therapy
  • Homologous replacement
  • Small DNA fragments

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics


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