Gene targeting of a CFTR allele in HT29 human epithelial cells.
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.
Montrose-Rafizadeh
C
C
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Kole
J
J
Bartkowski
L M
LM
Lee
L H
LH
Blackmon
D L
DL
Behnken
S E
SE
Gearhart
J D
JD
Cohn
J A
JA
Montrose
M H
MH
D000483:Alleles; D017398:Alternative Splicing / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis / Q000235:genetics*; D004847:Epithelial Cells; D005786:Gene Expression Regulation / Q000235:genetics; D005820:Genetic Testing; D005822:Genetic Vectors; D019073:HT29 Cells / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis / Q000502:physiology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator