Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells
- Univ. of North Carolina, Chapel Hill (United States)
- St. Mary's Hospital, London (England)
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of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters.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), possible passage number, influences the recovery of CFTR-targeted cells.
- OSTI ID:
- 5701708
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 88:23; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
EMBRYONIC CELLS
GENE RECOMBINATION
FIBROSIS
BIOLOGICAL MODELS
HEREDITARY DISEASES
ETIOLOGY
BACTERIOPHAGES
GENOME MUTATIONS
HERPES SIMPLEX
HUMAN POPULATIONS
MICE
PATIENTS
PHENYLALANINE
PHOSPHOTRANSFERASES
STEM CELLS
AMINO ACIDS
ANIMAL CELLS
ANIMALS
CARBOXYLIC ACIDS
DISEASES
ENZYMES
INFECTIOUS DISEASES
MAMMALS
MICROORGANISMS
MUTATIONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PATHOLOGICAL CHANGES
PHOSPHORUS-GROUP TRANSFERASES
POPULATIONS
PROTEINS
RODENTS
SKIN DISEASES
SOMATIC CELLS
TRANSFERASES
VERTEBRATES
VIRAL DISEASES
VIRUSES
550400* - Genetics