The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos
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{"title"=>"The expression of TALEN before fertilization provides a rapid knock-out phenotype in Xenopus laevis founder embryos", "type"=>"journal", "authors"=>[{"first_name"=>"Kei", "last_name"=>"Miyamoto", "scopus_author_id"=>"53363873800"}, {"first_name"=>"Ken Ichi T.", "last_name"=>"Suzuki", "scopus_author_id"=>"55503679400"}, {"first_name"=>"Miyuki", "last_name"=>"Suzuki", "scopus_author_id"=>"57111137100"}, {"first_name"=>"Yuto", "last_name"=>"Sakane", "scopus_author_id"=>"55953421800"}, {"first_name"=>"Tetsushi", "last_name"=>"Sakuma", "scopus_author_id"=>"36672079700"}, {"first_name"=>"Sarah", "last_name"=>"Herberg", "scopus_author_id"=>"56866060300"}, {"first_name"=>"Angela", "last_name"=>"Simeone", "scopus_author_id"=>"56381089500"}, {"first_name"=>"David", "last_name"=>"Simpson", "scopus_author_id"=>"54796975600"}, {"first_name"=>"Jerome", "last_name"=>"Jullien", "scopus_author_id"=>"15128954600"}, {"first_name"=>"Takashi", "last_name"=>"Yamamoto", "scopus_author_id"=>"55619316275"}, {"first_name"=>"J. B.", "last_name"=>"Gurdon", "scopus_author_id"=>"7102977003"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203 (Electronic)\r1932-6203 (Linking)", "pui"=>"608121414", "doi"=>"10.1371/journal.pone.0142946", "scopus"=>"2-s2.0-84957922305", "sgr"=>"84957922305", "issn"=>"19326203", "pmid"=>"26580070"}, "id"=>"00b8f3ea-8fa2-3ddd-b637-29f543edfb3b", "abstract"=>"Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.", "link"=>"http://www.mendeley.com/research/expression-talen-before-fertilization-provides-rapid-knockout-phenotype-xenopus-laevis-founder-embry", "reader_count"=>19, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Librarian"=>1, "Researcher"=>5, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1, "Student > Master"=>1, "Student > Bachelor"=>5}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Librarian"=>1, "Researcher"=>5, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1, "Student > Master"=>1, "Student > Bachelor"=>5}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>10, "Medicine and Dentistry"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}}, "reader_count_by_country"=>{"Argentina"=>1, "United States"=>1, "Japan"=>2}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/4442836"], "description"=>"<p>(A) Schematic diagram of the experiment to express TALENs in <i>Xenopus laevis</i> eggs before fertilization. (B) Development of <i>tyrosinase</i> (<i>tyr</i>) TALENs-expressed oocytes. TALEN mRNA-injected GV oocytes, followed by <i>in vitro</i> maturation and ICSI, were able to develop to the swimming tadpole stage although mRNA injection itself seems to decrease the developmental capacity of <i>in vitro</i> matured eggs. Actual numbers of embryos that were injected with sperm and that developed to each developmental stages are indicated next to the corresponding bars. (C-E) Expression of <i>tyr</i> TALENs in GV oocytes allowed almost complete albino phenotypes in F0 embryos and frogs. Enlarged pictures of two TALEN-RL-expressed tadpoles are shown in Fig 1D. Control R represents control embryos and frogs in which only right side TALEN was expressed.</p>", "links"=>[], "tags"=>["Xenopus laevis F 0 embryos", "genome editing", "intracytoplasmic sperm injection", "founder generation", "1 kb deletion", "Fertilization Provides", "host transfer", "Xenopus laevis Founder Embryos", "Xenopus laevis", "1 year", "F 0 generation", "model organism", "F 0 embryos", "Successful gene", "TALEN mRNA", "pigmentation gene tyrosinase", "injection methods", "GV oocytes", "judge phenotypes", "nuclease", "founder embryos"], "article_id"=>1608537, "categories"=>["Cell Biology", "Genetics", "Molecular Biology", "Evolutionary Biology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases"], "users"=>["Kei Miyamoto", "Ken-ichi T. Suzuki", "Miyuki Suzuki", "Yuto Sakane", "Tetsushi Sakuma", "Sarah Herberg", "Angela Simeone", "David Simpson", "Jerome Jullien", "Takashi Yamamoto", "J. B. Gurdon"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0142946.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Injection_of_TALEN_mRNAs_into_GV_oocytes_allows_efficient_tyrosinase_gene_disruption_/1608537", "title"=>"Injection of TALEN mRNAs into GV oocytes allows efficient <i>tyrosinase</i> gene disruption.", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-11-18 16:00:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/4442839"], "description"=>"<p>(A) Injection of <i>pax6</i> TALEN mRNAs to GV oocytes, followed by <i>in vitro</i> maturation and ICSI, recapitulated the null mutant phenotype of <i>pax6</i> in F0 embryos. Examples of knock-out tadpoles with different magnifications (a-f; <i>pax6</i> TALEN-expressed tadpoles, g-l; control tadpoles) are shown. The percentages of embryos that showed the knock-out phenotype of <i>pax6</i> are summarized in the graph, as judged by the degree of eye deformation. Embryos without TALEN mRNA injection are used as a control (Non injected). (B) Different phenotypes are observed at the tadpole stage between conventional embryo injection (Fig 2B) and the oocyte injection method (Fig 2A). TALEN mRNAs were injected into fertilized one-cell stage embryos. Examples of <i>pax6</i> TALEN-expressed tadpoles with different magnifications (a-d; <i>pax6</i> TALEN-expressed tadpoles, e-h; control tadpoles) are shown.</p>", "links"=>[], "tags"=>["Xenopus laevis F 0 embryos", "genome editing", "intracytoplasmic sperm injection", "founder generation", "1 kb deletion", "Fertilization Provides", "host transfer", "Xenopus laevis Founder Embryos", "Xenopus laevis", "1 year", "F 0 generation", "model organism", "F 0 embryos", "Successful gene", "TALEN mRNA", "pigmentation gene tyrosinase", "injection methods", "GV oocytes", "judge phenotypes", "nuclease", "founder embryos"], "article_id"=>1608539, "categories"=>["Cell Biology", "Genetics", "Molecular Biology", "Evolutionary Biology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases"], "users"=>["Kei Miyamoto", "Ken-ichi T. Suzuki", "Miyuki Suzuki", "Yuto Sakane", "Tetsushi Sakuma", "Sarah Herberg", "Angela Simeone", "David Simpson", "Jerome Jullien", "Takashi Yamamoto", "J. B. Gurdon"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0142946.g002"], "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotypes_of_embryos_that_were_injected_with_pax6_TALEN_mRNAs_/1608539", "title"=>"Phenotypes of embryos that were injected with <i>pax6</i> TALEN mRNAs.", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-11-18 16:00:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/4442848"], "description"=>"<p>(A) Double knock-out of <i>pax6</i> and <i>tyr</i> was achieved by TALEN mRNA injection to GV oocytes. A double knock-out tadpole is shown. (B) Detection of <i>pax6</i> and <i>tyr</i> mutants by RFLP analysis. Wild type embryos are cut by restriction enzymes (Ui: uninjected embryo), while mutant embryos are not digested. Digested PCR products appear at lower bands (marked by open arrowheads) and the undigested is marked by closed arrowheads. M represents 100 bp ladder marker. (C) Sequencing of three different embryos (#3, #6, #9; the numbers correspond to those in Fig 3B) revealed the 100% mutation rate in <i>pax6</i> and <i>tyr</i> double knock-out embryos.</p>", "links"=>[], "tags"=>["Xenopus laevis F 0 embryos", "genome editing", "intracytoplasmic sperm injection", "founder generation", "1 kb deletion", "Fertilization Provides", "host transfer", "Xenopus laevis Founder Embryos", "Xenopus laevis", "1 year", "F 0 generation", "model organism", "F 0 embryos", "Successful gene", "TALEN mRNA", "pigmentation gene tyrosinase", "injection methods", "GV oocytes", "judge phenotypes", "nuclease", "founder embryos"], "article_id"=>1608541, "categories"=>["Cell Biology", "Genetics", "Molecular Biology", "Evolutionary Biology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases"], "users"=>["Kei Miyamoto", "Ken-ichi T. Suzuki", "Miyuki Suzuki", "Yuto Sakane", "Tetsushi Sakuma", "Sarah Herberg", "Angela Simeone", "David Simpson", "Jerome Jullien", "Takashi Yamamoto", "J. B. Gurdon"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0142946.g003"], "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Double_knock_out_of_pax6_and_tyr_in_Xenopus_F0_embryos_/1608541", "title"=>"Double knock-out of <i>pax6</i> and <i>tyr</i> in <i>Xenopus</i> F0 embryos.", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-11-18 16:00:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/4442821", "https://ndownloader.figshare.com/files/4442824", "https://ndownloader.figshare.com/files/4442827", "https://ndownloader.figshare.com/files/4442830"], "description"=>"<div><p>Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in <i>Xenopus</i>, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by <i>in vitro</i> maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene <i>tyrosinase</i> and/or embryonic lethal gene <i>pax6</i> in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in <i>Xenopus laevis</i> F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in <i>Xenopus laevis</i>. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.</p></div>", "links"=>[], "tags"=>["Xenopus laevis F 0 embryos", "genome editing", "intracytoplasmic sperm injection", "founder generation", "1 kb deletion", "Fertilization Provides", "host transfer", "Xenopus laevis Founder Embryos", "Xenopus laevis", "1 year", "F 0 generation", "model organism", "F 0 embryos", "Successful gene", "TALEN mRNA", "pigmentation gene tyrosinase", "injection methods", "GV oocytes", "judge phenotypes", "nuclease", "founder embryos"], "article_id"=>1608545, "categories"=>["Cell Biology", "Genetics", "Molecular Biology", "Evolutionary Biology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases"], "users"=>["Kei Miyamoto", "Ken-ichi T. Suzuki", "Miyuki Suzuki", "Yuto Sakane", "Tetsushi Sakuma", "Sarah Herberg", "Angela Simeone", "David Simpson", "Jerome Jullien", "Takashi Yamamoto", "J. B. Gurdon"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0142946.s001", "https://dx.doi.org/10.1371/journal.pone.0142946.s002", "https://dx.doi.org/10.1371/journal.pone.0142946.s003", "https://dx.doi.org/10.1371/journal.pone.0142946.s004"], "stats"=>{"downloads"=>4, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_Expression_of_TALEN_before_Fertilization_Provides_a_Rapid_Knock_Out_Phenotype_in_Xenopus_laevis_Founder_Embryos/1608545", "title"=>"The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in <i>Xenopus laevis</i> Founder Embryos", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-11-18 16:00:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/4442851"], "description"=>"<p>(A) TALENs were designed to disrupt one of the exons of <i>mars2</i>-like (<i>mars2-l</i>) gene. PCR primers were used to confirm gene knock-out. Exons are marked by boxes. (B) The injection of TALEN mRNAs into GV oocytes (blue bar), but not into fertilized embryos (red bar), showed better knock-out of the <i>mars2-l</i> exon, as revealed by qPCR analyses. Control represents embryos injected only with right TALEN mRNA. N = 3–4 independent experiments. Error bars are standard errors. *P < 0.05. **P < 0.01. (C) Expression of TALENs before fertilization allowed the production of an embryo carrying only the deleted <i>mars2-l</i> exon in the F0 generation (lane 3, 7 and 8 in TALEN B-D, red color). Genomic DNA was extracted from single embryos at St. 10.5–11 and subjected to PCR analysis. M represents 1.5 kp and 100 bp ladder marker. (D) Large deletions of exon 2 in <i>mars-l</i> were confirmed by sequencing. Sample numbers correspond to those in Fig 4C. A part of inverted <i>mars-l</i> sequence was inserted in sample #3.</p>", "links"=>[], "tags"=>["Xenopus laevis F 0 embryos", "genome editing", "intracytoplasmic sperm injection", "founder generation", "1 kb deletion", "Fertilization Provides", "host transfer", "Xenopus laevis Founder Embryos", "Xenopus laevis", "1 year", "F 0 generation", "model organism", "F 0 embryos", "Successful gene", "TALEN mRNA", "pigmentation gene tyrosinase", "injection methods", "GV oocytes", "judge phenotypes", "nuclease", "founder embryos"], "article_id"=>1608543, "categories"=>["Cell Biology", "Genetics", "Molecular Biology", "Evolutionary Biology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases"], "users"=>["Kei Miyamoto", "Ken-ichi T. Suzuki", "Miyuki Suzuki", "Yuto Sakane", "Tetsushi Sakuma", "Sarah Herberg", "Angela Simeone", "David Simpson", "Jerome Jullien", "Takashi Yamamoto", "J. B. Gurdon"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0142946.g004"], "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_deletion_of_a_large_genetic_locus_is_achieved_by_the_expression_of_two_sets_of_TALENs_by_oocyte_injection_/1608543", "title"=>"The deletion of a large genetic locus is achieved by the expression of two sets of TALENs by oocyte injection.", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-11-18 16:00:09"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"11"}
  • {"unique-ip"=>"42", "full-text"=>"35", "pdf"=>"9", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"31", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"12"}
  • {"unique-ip"=>"39", "full-text"=>"26", "pdf"=>"20", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"17", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"1"}
  • {"unique-ip"=>"18", "full-text"=>"11", "pdf"=>"20", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"3"}
  • {"unique-ip"=>"29", "full-text"=>"10", "pdf"=>"23", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"4"}
  • {"unique-ip"=>"15", "full-text"=>"15", "pdf"=>"13", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"5"}
  • {"unique-ip"=>"11", "full-text"=>"7", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"6"}
  • {"unique-ip"=>"11", "full-text"=>"10", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"7"}
  • {"unique-ip"=>"9", "full-text"=>"7", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"8"}
  • {"unique-ip"=>"16", "full-text"=>"13", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"9"}
  • {"unique-ip"=>"4", "full-text"=>"3", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"11"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"12"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"1"}
  • {"unique-ip"=>"5", "full-text"=>"2", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"2"}
  • {"unique-ip"=>"7", "full-text"=>"5", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"3"}
  • {"unique-ip"=>"6", "full-text"=>"5", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"4"}
  • {"unique-ip"=>"11", "full-text"=>"11", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"5"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"6"}
  • {"unique-ip"=>"4", "full-text"=>"5", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"7"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"8"}
  • {"unique-ip"=>"12", "full-text"=>"10", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
  • {"unique-ip"=>"13", "full-text"=>"9", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"4", "cited-by"=>"1", "year"=>"2017", "month"=>"10"}
  • {"unique-ip"=>"15", "full-text"=>"12", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2017", "month"=>"11"}
  • {"unique-ip"=>"10", "full-text"=>"9", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
  • {"unique-ip"=>"7", "full-text"=>"8", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
  • {"unique-ip"=>"10", "full-text"=>"7", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
  • {"unique-ip"=>"3", "full-text"=>"2", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"1"}
  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"2", "full-text"=>"1", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"7", "full-text"=>"11", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"5"}
  • {"unique-ip"=>"7", "full-text"=>"4", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"8", "full-text"=>"6", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"6", "full-text"=>"6", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"6", "full-text"=>"6", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"4", "full-text"=>"3", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"2", "full-text"=>"3", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"8", "full-text"=>"7", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}

Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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