A method for the generation of conditional gene repair mutations in mice.
Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington's disease gene homolog (HDH:). Insertion of neostop within the HDH: intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous HDH: mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored HDH: expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.
Dragatsis
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Department of Genetics and Development and Department of Pathology, Columbia University, New York, NY 10032, USA.
Zeitlin
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D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods; D005838:Genotype; D000071058:Huntingtin Protein; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D014764:Viral Proteins
C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D014764:Viral Proteins; D012313:RNA; C045073:Cre recombinase; D019426:Integrases