Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells.
Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice with null alleles. Current methods of gene inactivation in ES cells introduce a neomycin gene (neo) cassette both as a mutagen and a selection marker for transfected cells. Although null alleles are valuable, changes at the nucleotide level of a gene are very important for functional analysis. One gene family in which subtle mutations would be particularly valuable are the clusters of Hox homeobox genes. Inactivation of gene in a cluster with a neo cassette that includes promoter/enhancer elements may deregulate transcription of neighbouring genes and generate a phenotype which is difficult to interpret. We describe here a highly efficient gene targeting method, termed the 'hit and run' procedure. This generates ES cells with subtle site-specific mutations with no selectable marker and may be useful for most genes. We have developed this procedure at the hypoxanthine phosphoribosyltransferase (hprt) locus and subsequently isolated ES cells with a premature stop codon in the homeobox of Hox-2.6 (ref. 14).
Hasty
P
P
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
Ramírez-Solis
R
R
Krumlauf
R
R
Bradley
A
A
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D004742:Enhancer Elements, Genetic; D005801:Genes, Homeobox; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism*; D014158:Transcription, Genetic
D007041:Hypoxanthine Phosphoribosyltransferase