Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.
AUTOR(ES)
Koller, B H
RESUMO
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of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters.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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=53004Documentos Relacionados
- Targeted mutation of the Hprt gene in mouse embryonic stem cells.
- A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis.
- Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.
- Medical genetics: advances in brief: Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts
- Cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts.
