Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.
A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possibly passage number, influences the recovery of CFTR-targeted cells.
Koller
B H
BH
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Kim
H S
HS
Latour
A M
AM
Brigman
K
K
Boucher
R C
RC
Scambler
P
P
Wainwright
B
B
Smithies
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D003062:Codon; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics; D004195:Disease Models, Animal; D004352:Drug Resistance, Microbial / Q000235:genetics; D004622:Embryo, Mammalian; D005091:Exons; D005821:Genetic Techniques; D019868:Kanamycin Kinase; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics; D013234:Stem Cells / Q000502:physiology*; D013937:Thymidine Kinase / Q000235:genetics
D003062:Codon; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D010770:Phosphotransferases; D013937:Thymidine Kinase; D019868:Kanamycin Kinase; D009355:Neomycin