Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA.
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.
Ando
Hideki
H
Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan. andoh@brain.riken.go.jp
Okamoto
Hitoshi
H
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*
C432678:6-bromo-4-diazomethyl-7-hydroxycoumarin; D001391:Azo Compounds; D003374:Coumarins; D012333:RNA, Messenger; D001616:beta-Galactosidase