Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos.
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.
Ando
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Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Furuta
T
T
Tsien
R Y
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Okamoto
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H
D000818:Animals; D001391:Azo Compounds / Q000737:chemistry / Q000494:pharmacology; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000008:administration & dosage / Q000737:chemistry / Q000528:radiation effects; D004625:Embryo, Nonmammalian / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005124:Eye Abnormalities / Q000139:chemically induced / Q000196:embryology / Q000473:pathology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects* / Q000528:radiation effects; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008845:Microinjections; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D009421:Nervous System Malformations / Q000139:chemically induced / Q000196:embryology / Q000473:pathology; D009990:Oryzias / Q000235:genetics; D010782:Photolysis; D016548:Prosencephalon / Q000002:abnormalities / Q000187:drug effects / Q000378:metabolism; D014176:Protein Biosynthesis / Q000528:radiation effects; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000737:chemistry / Q000528:radiation effects; D015533:Transcriptional Activation; D014466:Ultraviolet Rays; D015027:Zebrafish; D029961:Zebrafish Proteins; D000099266:Engrailed 2 Protein
D001391:Azo Compounds; D003374:Coumarins; D004247:DNA; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D029961:Zebrafish Proteins; C432678:6-bromo-4-diazomethyl-7-hydroxycoumarin; C079428:en2a protein, zebrafish; D000099266:Engrailed 2 Protein