title: Gene disruption/knock-in analysis of mONT3: vector construction by employing both in vivo and in vitro recombinations.
abstract: We report the isolation, spatial/temporal expression and gene disruption phenotype of the mouse ONT3 (mONT3) gene, which encodes a novel secreted signaling protein belonging to the Olfactomedin/Noelin/Tiarin family. During early embryogenesis, mONT3 is detected in the proximal region of the allantois on embryonic day (E) 7.25, in the lateral plate mesoderm on E 8.0 and in the CNS and heart on E 8.5. The homozygous mutant is born normal and fertile. For the expression pattern and loss-of-function analyses, we have successfully generated the LacZ-knock-in targeting vector directly from BACs carrying mouse genomic fragments by combining in vivo and in vitro recombination techniques. This approach enables rapid and reproducible construction of the fully functional vectors within two weeks without the use of restriction enzyme digestion and ligation, or the use of PCR-amplification of large genomic fragments. In addition, this method is applicable to rapid generation of transgenic vectors, demonstrating its versatility in reverse genetic studies.
authors.affiliation: Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe, Japan. mikeya@cdb.riken.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Ikeya
authors.forename: Masako
authors.initials: M
authors.lastname: Kawada
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nakazawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Sakuragi
authors.forename: Noriaki
authors.initials: N
authors.lastname: Sasai
authors.forename: Morio
authors.initials: M
authors.lastname: Ueno
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyonari
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakao
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Sasai
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D002642:Chick Embryo; D003433:Crosses, Genetic; D004622:Embryo, Mammalian / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics*; D005838:Genotype; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010802:Phylogeny; D011995:Recombination, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019076:Transgenes / Q000235:genetics*
chemical_list: D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C081451:olfactomedin