title: Production of conditional point mutant knockin mice.
abstract: Genetically engineered mice with point mutations in endogenous genes (i.e., knockin mice) are extremely useful tools for dissecting gene function. Currently available methodologies for creating knockin mice are limited in that the introduced mutation is globally present in all cells of the animal from conception through adulthood. In this report, we describe a strategy for creating mice in which a point mutant allele replaces the wild type allele in a conditional manner, e.g., in a tissue-specific and/or temporally restricted pattern. As proof of concept, we created mice that conditionally harbor a point mutated gamma-aminobutyric acid receptor subunit. In the absence of Cre recombinase, the engineered allele produces only wild type product with no evidence of expression of the mutant. In contrast, following Cre-mediated recombination, only the point mutant product is produced. By restricting Cre expression to subpopulations of neurons of postnatal animals, we demonstrate tissue-specific regulation of the point mutant knockin. This strategy will be useful for a wide variety of studies that require precise conditional replacement of an endogenous wild type gene with a point mutant.
authors.affiliation: Graduate Program in Biochemistry and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Kristen
authors.initials: K
authors.lastname: Skvorak
authors.forename: Bryce
authors.initials: B
authors.lastname: Vissel
authors.forename: Gregg E
authors.initials: GE
authors.lastname: Homanics
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D018390:Gene Targeting; D005838:Genotype; D019426:Integrases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism; D009928:Organ Specificity; D017354:Point Mutation; D011963:Receptors, GABA-A / Q000235:genetics*; D013997:Time Factors
chemical_list: C546241:Gabra1 protein, mouse; D011963:Receptors, GABA-A; C045073:Cre recombinase; D019426:Integrases