title: Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology.
abstract: Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx-based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene-trap mutagenesis. The SAGFLEx gene-trap cassette comprises the rabbit β-globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site-specific recombinases Cre and Flp. Insertion of the gene-trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene-trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial- and temporal-controlled manner.
authors.affiliation: Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Tatzberg, Dresden, 47-49, 01307, Germany.
authors.forename: Peggy
authors.initials: P
authors.lastname: Jungke
authors.affiliation: Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Tatzberg, Dresden, 47-49, 01307, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hans
authors.affiliation: Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Tatzberg, Dresden, 47-49, 01307, Germany.
authors.forename: Mansi
authors.initials: M
authors.lastname: Gupta
authors.affiliation: Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Tatzberg, Dresden, 47-49, 01307, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Machate
authors.affiliation: Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Tatzberg, Dresden, 47-49, 01307, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Zöller
authors.affiliation: Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Tatzberg, Dresden, 47-49, 01307, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Brand
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D003598:Cytoskeletal Proteins / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D004251:DNA Transposable Elements; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation; D015027:Zebrafish
chemical_list: D003598:Cytoskeletal Proteins; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C077570:Site-specific recombinase
keywords: FLEx switch; Lima1a; conditional alleles; zebrafish