title: Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system.
abstract: Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2(loxP/loxP) mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.
authors.affiliation: Department of Clinical Pharmacology, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. m-motoji@is.icc.u-tokai.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Motojima
authors.affiliation: Support Center for Medical Research and Education, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
authors.forename: Sanae
authors.initials: S
authors.lastname: Ogiwara
authors.affiliation: Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
authors.forename: Taiji
authors.initials: T
authors.lastname: Matsusaka
authors.affiliation: Department of Pathology, Rodent Genetic Engineering Core, Langone Medical Center, New York University, New York, NY, 10016, USA.
authors.forename: Sang Yong
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
authors.forename: Nobuho
authors.initials: N
authors.lastname: Sagawa
authors.affiliation: Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Abe
authors.affiliation: Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
mesh_terms: D000483:Alleles; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001761:Bleomycin / Q000494:pharmacology; D053595:Embryonic Stem Cells; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005091:Exons; D051858:Forkhead Transcription Factors / Q000172:deficiency* / Q000235:genetics; D018703:Founder Effect; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D005818:Genetic Engineering; D059765:Homologous Recombination; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics*; D008845:Microinjections; D009355:Neomycin / Q000494:pharmacology; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D045522:Recombinases / Q000235:genetics / Q000378:metabolism; D051437:Renal Insufficiency / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012641:Selection, Genetic; D014162:Transfection; D015053:Zygote / Q000187:drug effects / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C546029:Dre recombinase, E coli; D029968:Escherichia coli Proteins; C113731:Flp protein, bacteria; D051858:Forkhead Transcription Factors; D045522:Recombinases; C082078:mesenchyme fork head 1 protein; D001761:Bleomycin; C105427:Zeocin; D009355:Neomycin