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• {"link"=>{"url"=>"http://www.citeulike.org/user/natstreet/article/3001889"}, "post_time"=>"2008-07-15 11:09:18", "tag"=>"no-tag", "linkout"=>{"type"=>"DOI", "url"=>"http://dx.doi.org/10.1371/journal.pone.0001829"}, "username"=>"natstreet", "article_id"=>"3001889"}

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{"title"=>"Highly specific gene silencing by artificial miRNAs in rice", "type"=>"journal", "authors"=>[{"first_name"=>"Norman", "last_name"=>"Warthmann", "scopus_author_id"=>"6504376292"}, {"first_name"=>"Hao", "last_name"=>"Chen", "scopus_author_id"=>"57125292400"}, {"first_name"=>"Stephan", "last_name"=>"Ossowski", "scopus_author_id"=>"23489906200"}, {"first_name"=>"Detlef", "last_name"=>"Weigel", "scopus_author_id"=>"7006125954"}, {"first_name"=>"Philippe", "last_name"=>"Herve", "scopus_author_id"=>"24080304300"}], "year"=>2008, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"351936288", "sgr"=>"46649106719", "issn"=>"19326203", "pmid"=>"18350165", "scopus"=>"2-s2.0-46649106719", "doi"=>"10.1371/journal.pone.0001829", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)"}, "id"=>"8e162a5f-d4d2-376c-af46-cb70c9f71ab1", "abstract"=>"BACKGROUND:Endogenous microRNAs (miRNAs) are potent negative regulators of gene expression in plants and animals. Artificial miRNAs (amiRNAs)-designed to target one or several genes of interest-provide a new and highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants.\n\nMETHODOLOGY:We devised an amiRNA-based strategy for both japonica and indica type strains of cultivated rice, Oryza sativa. Using an endogenous rice miRNA precursor and customized 21mers, we designed amiRNA constructs targeting three different genes (Pds, Spl11, and Eui1/CYP714D1). Upon constitutive expression of these amiRNAs in the varieties Nipponbare (japonica) and IR64 (indica), the targeted genes are down-regulated by amiRNA-guided cleavage of the transcripts, resulting in the expected mutant phenotypes. The effects are highly specific to the target gene, the transgenes are stably inherited and they remain effective in the progeny.\n\nCONCLUSION/SIGNIFICANCE:Our results not only show that amiRNAs can efficiently trigger gene silencing in a monocot crop, but also that amiRNAs can effectively modulate agronomically important traits in varieties used in modern breeding programs. We provide all software tools and a protocol for the design of rice amiRNA constructs, which can be easily adapted to other crops. The approach is suited for candidate gene validation, comparative functional genomics between different varieties, and for improvement of agronomic performance and nutritional value.", "link"=>"http://www.mendeley.com/research/highly-specific-gene-silencing-artificial-mirnas-rice", "reader_count"=>311, "reader_count_by_academic_status"=>{"Unspecified"=>8, "Professor > Associate Professor"=>19, "Librarian"=>1, "Researcher"=>88, "Student > Doctoral Student"=>13, "Student > Ph. D. Student"=>103, "Student > Postgraduate"=>15, "Student > Master"=>27, "Other"=>8, "Student > Bachelor"=>9, "Lecturer"=>5, "Lecturer > Senior Lecturer"=>1, "Professor"=>14}, "reader_count_by_user_role"=>{"Unspecified"=>8, "Professor > Associate Professor"=>19, "Librarian"=>1, "Researcher"=>88, "Student > Doctoral Student"=>13, "Student > Ph. D. Student"=>103, "Student > Postgraduate"=>15, "Student > Master"=>27, "Other"=>8, "Student > Bachelor"=>9, "Lecturer"=>5, "Lecturer > Senior Lecturer"=>1, "Professor"=>14}, "reader_count_by_subject_area"=>{"Unspecified"=>13, "Engineering"=>1, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>19, "Agricultural and Biological Sciences"=>267, "Arts and Humanities"=>2, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Business, Management and Accounting"=>1, "Social Sciences"=>2, "Computer Science"=>3, "Economics, Econometrics and Finance"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Social Sciences"=>{"Social Sciences"=>2}, "Economics, Econometrics and Finance"=>{"Economics, Econometrics and Finance"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>267}, "Computer Science"=>{"Computer Science"=>3}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>19}, "Unspecified"=>{"Unspecified"=>13}, "Environmental Science"=>{"Environmental Science"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Arts and Humanities"=>{"Arts and Humanities"=>2}}, "reader_count_by_country"=>{"Colombia"=>1, "Argentina"=>2, "United States"=>7, "Philippines"=>4, "Japan"=>1, "Kenya"=>1, "Portugal"=>1, "Belgium"=>1, "Norway"=>2, "China"=>2, "Finland"=>1, "Poland"=>1, "Brazil"=>8, "Italy"=>1, "France"=>1, "Nigeria"=>1, "Chile"=>1, "Germany"=>3}, "group_count"=>13}

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• {"type"=>"COMMENT", "annotationUri"=>"info:doi/10.1371/annotation/7fb55435-5ba5-44e1-ad0f-1f9c4c2cd298", "title"=>"Referee Comments: Referee 1 (Andrew Cal)", "body"=>"Referee 1's Review (Andrew Cal):\n\nA great resource. The specificity of the spl11 knockdown was especially beautiful. Was there any correlation between copy number and efficacy of knock-down? Also, were all regenerates confirmed to have amiRNA sequence, or just the hpt gene? Partial insertion could skew your % of transgenic lines with efficient silencing downward. \nNice work.\n\n**********\nN.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements\n", "isRemoved"=>false, "created"=>"2008-03-21T16:58:29Z", "lastModified"=>"2008-03-21T16:58:29Z", "creator"=>{"userId"=>"80539"}, "highlightedText"=>"", "competingInterestStatement"=>{"creatorWasPrompted"=>false, "hasCompetingInterests"=>false}, "parentArticle"=>{"doi"=>"info:doi/10.1371/journal.pone.0001829", "state"=>"published", "journals"=>{"PLoSONE"=>{"journalKey"=>"PLoSONE", "eIssn"=>"1932-6203", "title"=>"PLOS ONE"}}}, "replyTreeSize"=>0, "mostRecentActivity"=>"2008-03-21T16:58:29Z", "replies"=>[]}
• {"type"=>"COMMENT", "annotationUri"=>"info:doi/10.1371/annotation/072dd7f5-d600-470b-b649-f6297c95ec5c", "title"=>"about vector IRS154", "body"=>"IRS154 is identical to \"pC1300intA.Ubi-tNos\". It was constructed by inserting a ubi promoter, first exon and first intron sequence (GenBank accession number: S94464,\nbasepair-positions 2-1993), multiple cloning site and nos polyadenylation sequence into pC1300intA (Ouwerkerk et al 2001, genbank accession number: AF294976), by Breitler and coworkers (unpubl.). Inquieries about this vector\nshould be directed to Pieter Ouwerkerk and/or E. Guiderdoni.", "isRemoved"=>false, "created"=>"2010-08-03T11:44:00Z", "lastModified"=>"2010-08-03T11:44:00Z", "creator"=>{"userId"=>"12119"}, "highlightedText"=>"", "competingInterestStatement"=>{"creatorWasPrompted"=>true, "hasCompetingInterests"=>false}, "parentArticle"=>{"doi"=>"info:doi/10.1371/journal.pone.0001829", "state"=>"published", "journals"=>{"PLoSONE"=>{"journalKey"=>"PLoSONE", "eIssn"=>"1932-6203", "title"=>"PLOS ONE"}}}, "replyTreeSize"=>0, "mostRecentActivity"=>"2010-08-03T11:44:00Z", "replies"=>[]}
• {"type"=>"COMMENT", "annotationUri"=>"info:doi/10.1371/annotation/0955cf51-9bca-42cb-91e3-9d0b141b3863", "title"=>"how to get NW55", "body"=>"NW55 is now available through www.addgene.org; on their website, search for plasmids associated with the keyword \"weigel\")", "isRemoved"=>false, "created"=>"2010-08-03T11:46:10Z", "lastModified"=>"2010-08-03T11:46:10Z", "creator"=>{"userId"=>"12119"}, "highlightedText"=>"", "competingInterestStatement"=>{"creatorWasPrompted"=>true, "hasCompetingInterests"=>false}, "parentArticle"=>{"doi"=>"info:doi/10.1371/journal.pone.0001829", "state"=>"published", "journals"=>{"PLoSONE"=>{"journalKey"=>"PLoSONE", "eIssn"=>"1932-6203", "title"=>"PLOS ONE"}}}, "replyTreeSize"=>0, "mostRecentActivity"=>"2010-08-03T11:46:10Z", "replies"=>[]}

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/935910"], "description"=>"<p>Cleavage site mapping was performed on mRNA from one transgenic plant for each transgene in both varieties (Nipponbare and IR64). Numbers above the arrows denote the number of clones ending at the particular position, with the total number of clones in parentheses. The binding energy (ΔG) of the RNA-RNA duplex between target (denoted by TIGR locus identifier) and amiRNA is given in kcal/mol and as a fraction of the calculated binding energy for a perfect match to the target site. Total RNA from two transgenic plants for each construct (leaf tissue for <i>SPl11</i> and <i>Pds</i>, young panicles for <i>Eui1</i>/<i>CYP714D1</i>) was used for RT-PCR for the target (histograms, top right), and small RNA blots (bottom right). Gel images are provided as loading control for small RNA blots. Comparison was to an empty vector control (IRS-154). Expression was normalized to the respective empty vector control. Error bars indicate the variation between technical replicates (range).</p>", "links"=>[], "tags"=>["characterization", "transgenic"], "article_id"=>606352, "categories"=>["Genetics", "Infectious Diseases", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0001829.g003", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Molecular_characterization_of_transgenic_plants_/606352", "title"=>"Molecular characterization of transgenic plants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2008-03-19 01:45:52"}
• {"files"=>["https://ndownloader.figshare.com/files/936104"], "description"=>"<p>Efficiency of different amiRNA constructs in Nipponbare and IR64 T₀ plants.</p>", "links"=>[], "tags"=>["amirna", "constructs", "nipponbare", "ir64"], "article_id"=>606544, "categories"=>["Genetics", "Infectious Diseases", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0001829.t002", "stats"=>{"downloads"=>1, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiency_of_different_amiRNA_constructs_in_Nipponbare_and_IR64_T_0_plants_/606544", "title"=>"Efficiency of different amiRNA constructs in Nipponbare and IR64 T₀ plants.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2008-03-19 01:49:04"}
• {"files"=>["https://ndownloader.figshare.com/files/460943", "https://ndownloader.figshare.com/files/460992", "https://ndownloader.figshare.com/files/461033", "https://ndownloader.figshare.com/files/461076", "https://ndownloader.figshare.com/files/461141", "https://ndownloader.figshare.com/files/461216", "https://ndownloader.figshare.com/files/461257", "https://ndownloader.figshare.com/files/461296", "https://ndownloader.figshare.com/files/461333", "https://ndownloader.figshare.com/files/461369", "https://ndownloader.figshare.com/files/461410", "https://ndownloader.figshare.com/files/461460"], "description"=>"<div><h3>Background</h3><p>Endogenous microRNAs (miRNAs) are potent negative regulators of gene expression in plants and animals. Artificial miRNAs (amiRNAs)–designed to target one or several genes of interest–provide a new and highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants.</p><h3>Methodology</h3><p>We devised an amiRNA-based strategy for both japonica and indica type strains of cultivated rice, <em>Oryza sativa</em>. Using an endogenous rice miRNA precursor and customized 21mers, we designed amiRNA constructs targeting three different genes (<em>Pds</em>, <em>Spl11,</em> and <em>Eui1/CYP714D1</em>). Upon constitutive expression of these amiRNAs in the varieties Nipponbare (japonica) and IR64 (indica), the targeted genes are down-regulated by amiRNA-guided cleavage of the transcripts, resulting in the expected mutant phenotypes. The effects are highly specific to the target gene, the transgenes are stably inherited and they remain effective in the progeny.</p><h3>Conclusion/Significance</h3><p>Our results not only show that amiRNAs can efficiently trigger gene silencing in a monocot crop, but also that amiRNAs can effectively modulate agronomically important traits in varieties used in modern breeding programs. We provide all software tools and a protocol for the design of rice amiRNA constructs, which can be easily adapted to other crops. The approach is suited for candidate gene validation, comparative functional genomics between different varieties, and for improvement of agronomic performance and nutritional value.</p></div>", "links"=>[], "tags"=>["mirnas"], "article_id"=>150837, "categories"=>["Genetics", "Cancer", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0001829.s001", "https://dx.doi.org/10.1371/journal.pone.0001829.s002", "https://dx.doi.org/10.1371/journal.pone.0001829.s003", "https://dx.doi.org/10.1371/journal.pone.0001829.s004", "https://dx.doi.org/10.1371/journal.pone.0001829.s005", "https://dx.doi.org/10.1371/journal.pone.0001829.s006", "https://dx.doi.org/10.1371/journal.pone.0001829.s007", "https://dx.doi.org/10.1371/journal.pone.0001829.s008", "https://dx.doi.org/10.1371/journal.pone.0001829.s009", "https://dx.doi.org/10.1371/journal.pone.0001829.s010", "https://dx.doi.org/10.1371/journal.pone.0001829.s011", "https://dx.doi.org/10.1371/journal.pone.0001829.s012"], "stats"=>{"downloads"=>53, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Highly_Specific_Gene_Silencing_by_Artificial_miRNAs_in_Rice/150837", "title"=>"Highly Specific Gene Silencing by Artificial miRNAs in Rice", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2008-03-19 00:13:57"}
• {"files"=>["https://ndownloader.figshare.com/files/936011"], "description"=>"<p>The program ‘RNAup’ <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001829#pone.0001829-Mckstein1\" target=\"_blank\">[33]</a> was used to assess the thermodynamics of RNA-RNA binding considering 80 bp surrounding the target site at 23°C. The predicted cleavage sites (between positions 10 and 11 of the miRNA) are highlighted in grey. ‘RNAup’ calculates the <i>effective</i> free hybridization energy (ΔG) as sum of the energies necessary to open the target site in the target mRNA (ΔGu_l), the internal folding of the amiRNA (ΔGu_s), and the energy gained from hybridization of amiRNA and target mRNA (ΔGint).</p>", "links"=>[], "tags"=>["amirna-mrna"], "article_id"=>606454, "categories"=>["Genetics", "Infectious Diseases", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0001829.g004", "stats"=>{"downloads"=>3, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Predicted_amiRNA_mRNA_interactions_/606454", "title"=>"Predicted amiRNA-mRNA interactions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2008-03-19 01:47:34"}
• {"files"=>["https://ndownloader.figshare.com/files/935456"], "description"=>"<p>Depicted is the secondary structure of the 255 bp derived from osa-MIR528 (245 bp plus the BamHI and KpNI restriction sites) as predicted by ‘RNAfold’ <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001829#pone.0001829-Mckstein1\" target=\"_blank\">[33]</a> (Vienna RNA package, <a href=\"http://www.tbi.univie.ac.at/RNA\" target=\"_blank\">http://www.tbi.univie.ac.at/RNA</a>) for 23°C. The sequences replaced in the different transgenes are boxed. The oligo designer in WMD2 <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001829#pone.0001829-Ossowski1\" target=\"_blank\">[25]</a> (<a href=\"http://wmd2.weigelworld.org\" target=\"_blank\">http://wmd2.weigelworld.org</a>) designs the amRNA* sequence (black) such that it pairs to the respective miRNA (red) in the same way as in osa-MIR528.</p>", "links"=>[], "tags"=>["osa-mir528", "stemloop"], "article_id"=>605897, "categories"=>["Genetics", "Infectious Diseases", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0001829.g001", "stats"=>{"downloads"=>2, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Secondary_Structure_of_the_osa_MIR528_stemloop_in_pNW55_/605897", "title"=>"Secondary Structure of the osa-MIR528 stemloop in pNW55.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2008-03-19 01:38:17"}
• {"files"=>["https://ndownloader.figshare.com/files/936067"], "description"=>"<p>Target genes and miRNA sequences.</p>", "links"=>[], "tags"=>["genes", "mirna"], "article_id"=>606510, "categories"=>["Genetics", "Infectious Diseases", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0001829.t001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Target_genes_and_miRNA_sequences_/606510", "title"=>"Target genes and miRNA sequences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2008-03-19 01:48:30"}
• {"files"=>["https://ndownloader.figshare.com/files/935724"], "description"=>"<p>The empty vector control is always on the left. (A–D) pNW76, construct targeting <i>Spl11</i>. (A) Nipponbare whole plants, (B) Nipponbare leaves, (C) IR64 whole plants, (D) IR64 leaves at maximum tillering stage. (E–H) pNW78, construct targeting <i>Pds</i>. (E) Nipponbare whole plants, (F) Nipponbare leaves, (G) IR64 whole plants, (H) IR64 leaves at tillering stage. (I–L) pNW81, construct targeting <i>Eui1</i>. (I) Nipponbare whole plants, (J) Nipponbare uppermost internode, (K) IR64 whole plants, (L) IR64 uppermost internode. The ruler in (J) and (L) is 60 cm long.</p>", "links"=>[], "tags"=>["plants", "overexpressing"], "article_id"=>606165, "categories"=>["Genetics", "Infectious Diseases", "Biotechnology", "Medicine"], "users"=>["Norman Warthmann", "Hao Chen", "Stephan Ossowski", "Detlef Weigel", "Philippe Hervé"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0001829.g002", "stats"=>{"downloads"=>2, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotypes_of_plants_overexpressing_amiRNAs_/606165", "title"=>"Phenotypes of plants overexpressing amiRNAs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2008-03-19 01:42:45"}

PMC Usage Stats | Further Information

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Relative Metric

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F1000Prime