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-...
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| Veröffentlicht in: | Nature genetics 2001-08, Vol.28 (4), p.317-325 |
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| creator | Okamoto, Hitoshi Ando, Hideki Furuta, Toshiaki Tsien, Roger Y |
| description | 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. |
| doi_str_mv | 10.1038/ng583 |
| format | Article |
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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.</description><identifier>ISSN: 1061-4036</identifier><identifier>EISSN: 1546-1718</identifier><identifier>DOI: 10.1038/ng583</identifier><identifier>PMID: 11479592</identifier><identifier>CODEN: NGENEC</identifier><language>eng</language><publisher>New York: Nature Publishing Group US</publisher><subject>Acids ; Agriculture ; Animal Genetics and Genomics ; Animals ; Azo Compounds - chemistry ; Azo Compounds - pharmacology ; Biological and medical sciences ; Biomedical and Life Sciences ; Biomedicine ; Brain research ; Cancer Research ; Coumarins - chemistry ; Coumarins - pharmacology ; DNA ; DNA - administration & dosage ; DNA - chemistry ; DNA - radiation effects ; Embryo, Nonmammalian - drug effects ; Embryo, Nonmammalian - metabolism ; Embryo, Nonmammalian - pathology ; Embryos ; Eye Abnormalities - chemically induced ; Eye Abnormalities - embryology ; Eye Abnormalities - pathology ; Fluorescence ; Fundamental and applied biological sciences. Psychology ; Gene expression ; Gene Expression Regulation, Developmental - drug effects ; Gene Expression Regulation, Developmental - radiation effects ; Gene Function ; Genes. Genome ; Genetics ; Green Fluorescent Proteins ; Homeodomain Proteins - biosynthesis ; Homeodomain Proteins - genetics ; Homeodomain Proteins - pharmacology ; Human Genetics ; Light ; Luminescent Proteins - biosynthesis ; Luminescent Proteins - genetics ; Microinjections ; Molecular and cellular biology ; Molecular genetics ; 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 ; new-technology ; Oryzias - genetics ; Phosphates ; Photolysis ; Physiological aspects ; Prosencephalon - abnormalities ; Prosencephalon - drug effects ; Prosencephalon - metabolism ; Protein Biosynthesis - radiation effects ; Publishing ; RNA ; RNA Stability - drug effects ; RNA, Messenger - administration & dosage ; RNA, Messenger - chemistry ; RNA, Messenger - radiation effects ; Science ; Sugar ; Transcriptional Activation ; Ultraviolet radiation ; Ultraviolet Rays ; Zebrafish</subject><ispartof>Nature genetics, 2001-08, Vol.28 (4), p.317-325</ispartof><rights>Springer Nature America, Inc. 2001</rights><rights>2002 INIST-CNRS</rights><rights>COPYRIGHT 2001 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Aug 2001</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c626t-28457f469e5fe819be0445c6f816220334c7914cd78cfdffab69e06a6bfe16f43</citedby><cites>FETCH-LOGICAL-c626t-28457f469e5fe819be0445c6f816220334c7914cd78cfdffab69e06a6bfe16f43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/ng583$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/ng583$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,2727,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=14151494$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11479592$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Okamoto, Hitoshi</creatorcontrib><creatorcontrib>Ando, Hideki</creatorcontrib><creatorcontrib>Furuta, Toshiaki</creatorcontrib><creatorcontrib>Tsien, Roger Y</creatorcontrib><title>Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos</title><title>Nature genetics</title><addtitle>Nat Genet</addtitle><addtitle>Nat Genet</addtitle><description>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.</description><subject>Acids</subject><subject>Agriculture</subject><subject>Animal Genetics and Genomics</subject><subject>Animals</subject><subject>Azo Compounds - chemistry</subject><subject>Azo Compounds - pharmacology</subject><subject>Biological and medical sciences</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Brain research</subject><subject>Cancer Research</subject><subject>Coumarins - chemistry</subject><subject>Coumarins - pharmacology</subject><subject>DNA</subject><subject>DNA - administration & dosage</subject><subject>DNA - chemistry</subject><subject>DNA - radiation effects</subject><subject>Embryo, Nonmammalian - drug effects</subject><subject>Embryo, Nonmammalian - metabolism</subject><subject>Embryo, Nonmammalian - pathology</subject><subject>Embryos</subject><subject>Eye Abnormalities - chemically induced</subject><subject>Eye Abnormalities - embryology</subject><subject>Eye Abnormalities - pathology</subject><subject>Fluorescence</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene expression</subject><subject>Gene Expression Regulation, Developmental - drug effects</subject><subject>Gene Expression Regulation, Developmental - radiation effects</subject><subject>Gene Function</subject><subject>Genes. Genome</subject><subject>Genetics</subject><subject>Green Fluorescent Proteins</subject><subject>Homeodomain Proteins - biosynthesis</subject><subject>Homeodomain Proteins - genetics</subject><subject>Homeodomain Proteins - pharmacology</subject><subject>Human Genetics</subject><subject>Light</subject><subject>Luminescent Proteins - biosynthesis</subject><subject>Luminescent Proteins - genetics</subject><subject>Microinjections</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Nerve Tissue Proteins - biosynthesis</subject><subject>Nerve Tissue Proteins - genetics</subject><subject>Nerve Tissue Proteins - pharmacology</subject><subject>Nervous System Malformations - chemically induced</subject><subject>Nervous System Malformations - embryology</subject><subject>Nervous System Malformations - pathology</subject><subject>new-technology</subject><subject>Oryzias - genetics</subject><subject>Phosphates</subject><subject>Photolysis</subject><subject>Physiological aspects</subject><subject>Prosencephalon - abnormalities</subject><subject>Prosencephalon - drug effects</subject><subject>Prosencephalon - metabolism</subject><subject>Protein Biosynthesis - radiation effects</subject><subject>Publishing</subject><subject>RNA</subject><subject>RNA Stability - drug effects</subject><subject>RNA, Messenger - administration & dosage</subject><subject>RNA, Messenger - chemistry</subject><subject>RNA, Messenger - radiation effects</subject><subject>Science</subject><subject>Sugar</subject><subject>Transcriptional Activation</subject><subject>Ultraviolet radiation</subject><subject>Ultraviolet Rays</subject><subject>Zebrafish</subject><issn>1061-4036</issn><issn>1546-1718</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNqNkdFL3TAYxYNMplP_gsEokwk-VJM2TdPHi5tOEB1u-hrS9EuN3CbXJB1zf_1y18suV_YgeUjI-X3nkByEDgg-Ibjkp7aveLmFdklFWU5qwt-kM2Ykp7hkO-hdCI8YE0oxf4t2CKF1UzXFLrr49uCiywfojIzQZT1YyKSK5qeMxtlsDMb2mZJ90m6vZ6efr2eZsdlvaL3UJjxkMLT-2YV9tK3lPMDBat9Dd-dffpx9za9uLi7PZle5YgWLecFpVWvKGqg0cNK0gCmtFNOcsKLAZUlV3RCqupor3Wkt24RiJlmrgTBNyz30cfJdePc0Qoji0Y3epkhRFAVjvKpZgg4nqJdzEMZqF71UgwlKzAgvack4XVqd_IdKq4PBKGdBm3S_MXC8MZCYCL9iL8cQxOX329ezN_eb7KeJVd6F4EGLhTeD9M-CYLEsV_wtN3EfVm8f21TYmlq1uTZayKDkXHtplQlrjpKK0GYZeDRxIUm2B7_-xJeJ7yfQyjh6-Oc0qX8AgHG9EA</recordid><startdate>20010801</startdate><enddate>20010801</enddate><creator>Okamoto, Hitoshi</creator><creator>Ando, Hideki</creator><creator>Furuta, Toshiaki</creator><creator>Tsien, Roger Y</creator><general>Nature Publishing Group US</general><general>Nature Publishing Group</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M7N</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope></search><sort><creationdate>20010801</creationdate><title>Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos</title><author>Okamoto, Hitoshi ; Ando, Hideki ; Furuta, Toshiaki ; Tsien, Roger Y</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c626t-28457f469e5fe819be0445c6f816220334c7914cd78cfdffab69e06a6bfe16f43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Acids</topic><topic>Agriculture</topic><topic>Animal Genetics and Genomics</topic><topic>Animals</topic><topic>Azo Compounds - chemistry</topic><topic>Azo Compounds - pharmacology</topic><topic>Biological and medical sciences</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Brain research</topic><topic>Cancer Research</topic><topic>Coumarins - chemistry</topic><topic>Coumarins - pharmacology</topic><topic>DNA</topic><topic>DNA - administration & dosage</topic><topic>DNA - chemistry</topic><topic>DNA - radiation effects</topic><topic>Embryo, Nonmammalian - drug effects</topic><topic>Embryo, Nonmammalian - metabolism</topic><topic>Embryo, Nonmammalian - pathology</topic><topic>Embryos</topic><topic>Eye Abnormalities - chemically induced</topic><topic>Eye Abnormalities - embryology</topic><topic>Eye Abnormalities - pathology</topic><topic>Fluorescence</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene expression</topic><topic>Gene Expression Regulation, Developmental - drug effects</topic><topic>Gene Expression Regulation, Developmental - radiation effects</topic><topic>Gene Function</topic><topic>Genes. Genome</topic><topic>Genetics</topic><topic>Green Fluorescent Proteins</topic><topic>Homeodomain Proteins - biosynthesis</topic><topic>Homeodomain Proteins - genetics</topic><topic>Homeodomain Proteins - pharmacology</topic><topic>Human Genetics</topic><topic>Light</topic><topic>Luminescent Proteins - biosynthesis</topic><topic>Luminescent Proteins - genetics</topic><topic>Microinjections</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Nerve Tissue Proteins - biosynthesis</topic><topic>Nerve Tissue Proteins - genetics</topic><topic>Nerve Tissue Proteins - pharmacology</topic><topic>Nervous System Malformations - chemically induced</topic><topic>Nervous System Malformations - embryology</topic><topic>Nervous System Malformations - pathology</topic><topic>new-technology</topic><topic>Oryzias - genetics</topic><topic>Phosphates</topic><topic>Photolysis</topic><topic>Physiological aspects</topic><topic>Prosencephalon - abnormalities</topic><topic>Prosencephalon - drug effects</topic><topic>Prosencephalon - metabolism</topic><topic>Protein Biosynthesis - radiation effects</topic><topic>Publishing</topic><topic>RNA</topic><topic>RNA Stability - drug effects</topic><topic>RNA, Messenger - administration & dosage</topic><topic>RNA, Messenger - chemistry</topic><topic>RNA, Messenger - radiation effects</topic><topic>Science</topic><topic>Sugar</topic><topic>Transcriptional Activation</topic><topic>Ultraviolet radiation</topic><topic>Ultraviolet Rays</topic><topic>Zebrafish</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Okamoto, Hitoshi</creatorcontrib><creatorcontrib>Ando, Hideki</creatorcontrib><creatorcontrib>Furuta, Toshiaki</creatorcontrib><creatorcontrib>Tsien, Roger Y</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE 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Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><jtitle>Nature genetics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Okamoto, Hitoshi</au><au>Ando, Hideki</au><au>Furuta, Toshiaki</au><au>Tsien, Roger Y</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos</atitle><jtitle>Nature genetics</jtitle><stitle>Nat Genet</stitle><addtitle>Nat Genet</addtitle><date>2001-08-01</date><risdate>2001</risdate><volume>28</volume><issue>4</issue><spage>317</spage><epage>325</epage><pages>317-325</pages><issn>1061-4036</issn><eissn>1546-1718</eissn><coden>NGENEC</coden><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.</abstract><cop>New York</cop><pub>Nature Publishing Group US</pub><pmid>11479592</pmid><doi>10.1038/ng583</doi><tpages>9</tpages></addata></record> |
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| ispartof | Nature genetics, 2001-08, Vol.28 (4), p.317-325 |
| issn | 1061-4036 1546-1718 |
| language | eng |
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| source | MEDLINE; Nature; SpringerLink Journals - AutoHoldings |
| subjects | Acids Agriculture Animal Genetics and Genomics Animals Azo Compounds - chemistry Azo Compounds - pharmacology Biological and medical sciences Biomedical and Life Sciences Biomedicine Brain research Cancer Research Coumarins - chemistry Coumarins - pharmacology DNA DNA - administration & dosage DNA - chemistry DNA - radiation effects Embryo, Nonmammalian - drug effects Embryo, Nonmammalian - metabolism Embryo, Nonmammalian - pathology Embryos Eye Abnormalities - chemically induced Eye Abnormalities - embryology Eye Abnormalities - pathology Fluorescence Fundamental and applied biological sciences. Psychology Gene expression Gene Expression Regulation, Developmental - drug effects Gene Expression Regulation, Developmental - radiation effects Gene Function Genes. Genome Genetics Green Fluorescent Proteins Homeodomain Proteins - biosynthesis Homeodomain Proteins - genetics Homeodomain Proteins - pharmacology Human Genetics Light Luminescent Proteins - biosynthesis Luminescent Proteins - genetics Microinjections Molecular and cellular biology Molecular genetics 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 new-technology Oryzias - genetics Phosphates Photolysis Physiological aspects Prosencephalon - abnormalities Prosencephalon - drug effects Prosencephalon - metabolism Protein Biosynthesis - radiation effects Publishing RNA RNA Stability - drug effects RNA, Messenger - administration & dosage RNA, Messenger - chemistry RNA, Messenger - radiation effects Science Sugar Transcriptional Activation Ultraviolet radiation Ultraviolet Rays Zebrafish |
| title | Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos |
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