Gene targeting of a CFTR allele in HT29 human epithelial cells.

Published

Journal Article

HT29 cells endogenously express the cystic fibrosis transmembrane conductance regulator (CFTR) and have been used previously as a model to examine cellular regulation of CFTR expression and chloride secretory function. Homologous recombination has been used to specifically disrupt CFTR transcription in the HT29-18-C1 subclone. Experiments demonstrate successful disruption of a CFTR allele by DNA constructs, which target insertion of the neomycin phosphotransferase gene into CFTR exon 1 via homologous recombination. The mutation of one allele is a partial knockout because this cell line has multiple CFTR alleles. The mutation is confirmed by polymerase chain reaction (PCR) and genomic Southern blot analysis. A 52-68% reduction in CFTR mRNA levels is observed in the mutant cell line by both Northern and PCR analysis. However, Western blots show no decrease in total CFTR protein levels. Consistent with the lack of reduction in CFTR protein, the partial knockout mutant does not demonstrate alterations in cyclic AMP or calcium stimulation of chloride efflux or net osmolyte loss. Results suggest that posttranscriptional regulation of CFTR levels may contribute to maintenance of cellular chloride transport function.

Full Text

Duke Authors

Cited Authors

  • Montrose-Rafizadeh, C; Kole, J; Bartkowski, LM; Lee, LH; Blackmon, DL; Behnken, SE; Gearhart, JD; Cohn, JA; Montrose, MH

Published Date

  • March 1997

Published In

Volume / Issue

  • 170 / 3

Start / End Page

  • 299 - 308

PubMed ID

  • 9066787

Pubmed Central ID

  • 9066787

Electronic International Standard Serial Number (EISSN)

  • 1097-4652

International Standard Serial Number (ISSN)

  • 0021-9541

Digital Object Identifier (DOI)

  • 10.1002/(sici)1097-4652(199703)170:3<299::aid-jcp11>3.0.co;2-a

Language

  • eng