PUBLICATION

Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos

Authors
Ando, H., Furuta, T., Tsien, R.Y., and Okamoto, H.
ID
ZDB-PUB-010807-5
Date
2001
Source
Nature Genetics   28(4): 317-325 (Journal)
Registered Authors
Ando, Hideki
Keywords
none
MeSH Terms
  • Animals
  • Azo Compounds/chemistry
  • Azo Compounds/pharmacology
  • Coumarins/chemistry
  • Coumarins/pharmacology
  • DNA/administration & dosage
  • DNA/chemistry
  • DNA/radiation effects
  • Embryo, Nonmammalian/drug effects*
  • Embryo, Nonmammalian/metabolism
  • Embryo, Nonmammalian/pathology
  • Eye Abnormalities/chemically induced
  • Eye Abnormalities/embryology
  • Eye Abnormalities/pathology
  • Gene Expression Regulation, Developmental/drug effects*
  • Gene Expression Regulation, Developmental/radiation effects
  • Green Fluorescent Proteins
  • Homeodomain Proteins/biosynthesis
  • Homeodomain Proteins/genetics
  • Homeodomain Proteins/pharmacology
  • Luminescent Proteins/biosynthesis
  • Luminescent Proteins/genetics
  • Microinjections
  • Nerve Tissue Proteins/biosynthesis
  • Nerve Tissue Proteins/genetics
  • Nerve Tissue Proteins/pharmacology
  • Nervous System Malformations/chemically induced
  • Nervous System Malformations/embryology
  • Nervous System Malformations/pathology
  • Oryzias/genetics
  • Photolysis*
  • Prosencephalon/abnormalities
  • Prosencephalon/drug effects
  • Prosencephalon/metabolism
  • Protein Biosynthesis/radiation effects
  • RNA Stability/drug effects
  • RNA, Messenger/administration & dosage*
  • RNA, Messenger/chemistry
  • RNA, Messenger/radiation effects
  • Transcriptional Activation
  • Ultraviolet Rays
  • Zebrafish
PubMed
11479592 Full text @ Nat. Genet.
Abstract
We report a new and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos as an alternative to the gene 'knockdown' approach using antisense, morpholino-modified oligonucleotides (morpholinos). The synthetic compound 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo) forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA, a process known as caging. The 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group binds to approximately 30 sites on the phosphate moieties per 1 kb of RNA sequence. Bhc-caged mRNA undergoes photolysis (uncaging) when exposed to long-wave ultraviolet light (350 to 365 nm). We show that Bhc-caged green fluorescent protein (Gfp) mRNA has severely reduced translational activity in vitro, whereas illumination of Bhc-caged mRNA with ultraviolet light leads to partial recovery of translational activity. Bhc-caged mRNA is highly stable in zebrafish embryos. In embryos injected with Bhc-caged Gfp mRNA at the one-cell stage, GFP protein expression and fluorescence is specifically induced by ultraviolet light. We also show that, consistent with results obtained using other methods, uncaging eng2a (which encodes the transcription factor Engrailed2a) in the head region during early development causes a severe reduction in the size of the eye and enhanced development of the midbrain and the midbrain-hindbrain boundary at the expense of the forebrain.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping