ZFIN ID: ZDB-PUB-031229-5
Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA
Ando, H. and Okamoto, H.
Date: 2003
Source: Methods in cell science : an official journal of the Society for In Vitro Biology 25(1-2): 25-31 (Review)
Registered Authors: Ando, Hideki, Okamoto, Hitoshi
Keywords: Bhc-diazo, Caged mRNA, Caging, Ectopic gene expression, Zebrafish
MeSH Terms:
  • Animals
  • Azo Compounds/chemistry*
  • Cloning, Molecular
  • Coumarins/chemistry*
  • Embryo, Nonmammalian/anatomy & histology
  • Microinjections
  • Photolysis/radiation effects*
  • RNA, Messenger/genetics
  • RNA, Messenger/metabolism*
  • Ultraviolet Rays
  • Zebrafish/anatomy & histology
  • Zebrafish/genetics*
  • beta-Galactosidase/genetics
  • beta-Galactosidase/metabolism*
PubMed: 14739584 Full text @ Methods Cell Sci.
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 (350~365 nm). In this paper we describe the technique and detailed procedures for spatially and temporally controlled induction of gene expression in zebrafish embryos.