Ando, H. and Okamoto, H. (2003) Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA. Methods in cell science : an official journal of the Society for In Vitro Biology. 25(1-2):25-31.
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.