title: Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA.
abstract: We previously reported mRNA caging technology as a novel and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos and as an alternative to the 'gene knockdown' approach using antisense morpholino oligonucleotides. The caging reagent used is 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo), which forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA. Mainly because of the reduced solubility of caged mRNA in aqueous solutions, special care in handling is needed. The Bhc-diazo group binds to the phosphate moieties of RNA and abolishes the translational activity of the latter. The translational activity of Bhc-caged mRNA is restored by photolysis/uncaging when exposed to long-wave UV light (350 approximately 365 nm). In this paper we describe the technique and detailed procedures for spatially and temporally controlled induction of gene expression in zebrafish embryos.
authors.affiliation: Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan. andoh@brain.riken.go.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Ando
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Okamoto
mesh_terms: D000818:Animals; D001391:Azo Compounds / Q000737:chemistry*; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry*; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology; D008845:Microinjections; D010782:Photolysis / Q000528:radiation effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000033:anatomy & histology / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: C432678:6-bromo-4-diazomethyl-7-hydroxycoumarin; D001391:Azo Compounds; D003374:Coumarins; D012333:RNA, Messenger; D001616:beta-Galactosidase