Disruption of the cystic fibrosis transmembrane conductance regulator gene in embryonic stem cells by gene targeting |
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Authors: | Rosemary Ratcliff Martin J. Evans Joanne Doran Brandon J. Wainwaright Robert Williamson William H. Colledge |
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Affiliation: | (1) Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, CB2 1QR Cambridge, UK;(2) Department of Biochemistry and Molecular Genetics, St Mary’s Hospital Medical School, Norfolk Place, W2 1PG London, UK |
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Abstract: | We have successfully disrupted thecftr (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 functionalhprt (hypoxanthine phosphoribosyl transferase) mini-gene into exon 10 of thecftr gene. Targeted embryonic stem cell clones were identified by PCR screening and confirmed by Southern blot analysis. One of thecftr 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 thehprt gene in exon 10 with the concomitant introduction of the ΔF508 mutation or other mutations. |
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Keywords: | Cftr embryonic stem cells gene targeting homologous recombination hprt |
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