Disruption of the cystic fibrosis transmembrane conductance regulator gene in embryonic stem cells by gene targeting.
We have successfully disrupted the cftr (cystic fibrosis transmembrane conductance regulator) gene at its endogenous locus in embryonic stem cells by gene targeting. We are using a double replacement strategy to introduce subtle mutations into exon 10. We report here the first step of creating a null mutation by insertion of a functional hprt (hypoxanthine phosphoribosyl transferase) mini-gene into exon 10 of the cftr gene. Targeted embryonic stem cell clones were identified by PCR screening and confirmed by Southern blot analysis. One of the cftr targeted clones has been injected into recipient blastocysts and shown to contribute to chimaeras. The targeted clones will now be used as the starting point for a second gene targeting step to remove the hprt gene in exon 10 with the concomitant introduction of the delta F508 mutation or other mutations.
Ratcliff
R
R
Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, UK.
Evans
M J
MJ
Doran
J
J
Wainwright
B J
BJ
Williamson
R
R
Colledge
W H
WH
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000502:physiology*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004622:Embryo, Mammalian / Q000502:physiology; D005091:Exons; D005260:Female; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D013234:Stem Cells / Q000502:physiology*
C505032:CFTR protein, human; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D007041:Hypoxanthine Phosphoribosyltransferase