Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA

Authors: Ando H.¹; Okamoto H.²

Source: Methods in Cell Science, Volume 25, Numbers 1-2, 2003 , pp. 25-31(7)

Publisher: Springer

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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 sim

365 nm). In this paper we describe the technique and detailed procedures for spatially and temporally controlled induction of gene expression in zebrafish embryos.

Keywords: Bhc-diazo; Caged mRNA; Caging; Ectopic gene expression; Zebrafish

Document Type: Research article

Affiliations: 1: Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan, Email: andoh@brain.riken.go.jp 2: Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 3-4-5 Nihonba

Publication date: 2003-01-01