System for Stable β-Estradiol-Inducible Gene Expression in the Moss Physcomitrella patens
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{"title"=>"System for Stable β-Estradiol-Inducible Gene Expression in the Moss Physcomitrella patens", "type"=>"journal", "authors"=>[{"first_name"=>"Minoru", "last_name"=>"Kubo", "scopus_author_id"=>"57200196428"}, {"first_name"=>"Akihiro", "last_name"=>"Imai", "scopus_author_id"=>"36838672100"}, {"first_name"=>"Tomoaki", "last_name"=>"Nishiyama", "scopus_author_id"=>"7202639921"}, {"first_name"=>"Masaki", "last_name"=>"Ishikawa", "scopus_author_id"=>"56843329300"}, {"first_name"=>"Yoshikatsu", "last_name"=>"Sato", "scopus_author_id"=>"7410374103"}, {"first_name"=>"Tetsuya", "last_name"=>"Kurata", "scopus_author_id"=>"8983983300"}, {"first_name"=>"Yuji", "last_name"=>"Hiwatashi", "scopus_author_id"=>"22947323900"}, {"first_name"=>"Ralf", "last_name"=>"Reski", "scopus_author_id"=>"7003308361"}, {"first_name"=>"Mitsuyasu", "last_name"=>"Hasebe", "scopus_author_id"=>"7005059775"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24086772", "doi"=>"10.1371/journal.pone.0077356", "issn"=>"19326203", "scopus"=>"2-s2.0-84884694684", "pui"=>"369903739", "sgr"=>"84884694684"}, "id"=>"f96dee48-93d9-36c3-9cb7-9e80f24065e5", "abstract"=>"Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.", "link"=>"http://www.mendeley.com/research/system-stable-%CE%B2estradiolinducible-gene-expression-moss-physcomitrella-patens", "reader_count"=>55, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>16, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>12, "Student > Postgraduate"=>2, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>9, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>16, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>12, "Student > Postgraduate"=>2, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>9, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_subject_area"=>{"Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>13, "Agricultural and Biological Sciences"=>41}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>41}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>13}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Czech Republic"=>1, "United States"=>1, "Brazil"=>2, "Italy"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1218706"], "description"=>"<p>Schematic representations of pER8 (A: modified from [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B22\" target=\"_blank\">22</a>]), pPGX (B), pLGZ2 (C) vectors. RB: right border [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B22\" target=\"_blank\">22</a>], PG10-90: a G10-90 synthetic promoter [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B32\" target=\"_blank\">32</a>], TrbcSE9: a Pea rbcS E9 terminator [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B33\" target=\"_blank\">33</a>], Pnos: Agrobacterium nopaline synthase promoter [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B17\" target=\"_blank\">17</a>], <i>HPT</i>: the hygromycin phosphotransferase gene [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B17\" target=\"_blank\">17</a>], Tnos, an agrobacterium nopaline synthase terminator [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B17\" target=\"_blank\">17</a>], LexA:Pm35S: eight copies of LexA operators [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B22\" target=\"_blank\">22</a>] connected to CaMV minimal 35S promoter [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B23\" target=\"_blank\">23</a>], MCS: multi cloning site including <i>Xho</i>I, <i>Asc</i>I, <i>Apa</i>I, <i>Pac</i>I, and <i>Spe</i>I. TrbcS: a TrbcS terminator [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B34\" target=\"_blank\">34</a>], LB: left border [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B22\" target=\"_blank\">22</a>], PIG1R and PIG1L: DNA fragments for homologous recombination to the PIG1 putative neutral site [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B14\" target=\"_blank\">14</a>], GW: the Gateway cassette rfA containing the <i>chloramphenicol </i><i>resistance</i> gene and the <i>ccdB</i> gene flanked by attR1 and attR2 sites (Life Technologies), Tpea3A: a Tpea3A terminator [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B34\" target=\"_blank\">34</a>], PGX: one of GX promoters, <i>XVE</i>: the chimeric gene with a LexA-binding [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B28\" target=\"_blank\">28</a>,<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B29\" target=\"_blank\">29</a>], a VP16 activator [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B30\" target=\"_blank\">30</a>], and an estrogen receptor domain [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B31\" target=\"_blank\">31</a>], aphIV: the hygromicin phosphotransferase expression cassette [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B35\" target=\"_blank\">35</a>,<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B36\" target=\"_blank\">36</a>], PTA2R and PTA2L: DNA fragments targeting to the PTA2 putative neutral site, loxP: the sequences for site-specific recombination by Cre recombinase [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B55\" target=\"_blank\">55</a>], zeo: the bleomycin resistance protein expression cassette [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B36\" target=\"_blank\">36</a>]. Backbone of pER8 is pZP200 [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B22\" target=\"_blank\">22</a>]. Backbones of pPGX and pLGZ2 are pBluescriptII [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#B49\" target=\"_blank\">49</a>].</p>", "links"=>[], "tags"=>["xve"], "article_id"=>810268, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_representation_of_the_XVE_system_/810268", "title"=>"Schematic representation of the XVE system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218707"], "description"=>"<p>Fluorescence images of protonemata (A) and gametophores (B) of GX6-NGG#63 and GX8-NGG#4 lines. Fluorescent signals were observed after 24 h inoculation in water with (+) or without (-) 1 µM β-estradiol. Yellowish color of leaf veins in GX8-NGG#4 is caused by reflection but not by NGG signals. (C) Magnified views of apexes of gametophores in GX6-NGG#63 and GX8-NGG#4 lines. Pictures in A, B, and C panels are at the same magnification. Bars = 100 µm.</p>", "links"=>[], "tags"=>["patterns", "ngg", "induced", "xve"], "article_id"=>810269, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Spatial_expression_patterns_of_NGG_induced_by_P_patens_XVE_system_/810269", "title"=>"Spatial expression patterns of NGG induced by <i>P</i><i>. patens</i> XVE system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218708"], "description"=>"<p>(A, B) Transcript amounts of <i>NGG</i> (A) and <i>TUA1</i> (B) in protonemata incubated for 8 days after propagation. Three independently transformed lines harboring a single copy of each DNA fragment at the PIG1 targeting site were analyzed (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077356#pone.0077356.s004\" target=\"_blank\">Figures S4 to S6</a>). Copy numbers of the <i>NGG</i> and <i>TUA1</i> transcripts per pg total RNA were estimated by RT-qPCR. In HSP:NGG lines, protonemata were cultured for 8 days at 25°C and collected after 1 h incubation at 25°C (crosses) or 38°C (squares).</p>", "links"=>[], "tags"=>["transcripts"], "article_id"=>810270, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Amounts_of_NGG_and_TUA1_transcripts_in_Pm35S_NGG_Pact1_NGG_and_HSP_NGG_lines_/810270", "title"=>"Amounts of <i>NGG</i> and <i>TUA1</i> transcripts in Pm35S:NGG, Pact1:NGG, and HSP:NGG lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218709"], "description"=>"<p>(A, B) Transcript amounts of <i>NGG</i> (A) and <i>TUA1</i> (B) in protonemata immersed in water with (+: circles) or without (-: crosses) 1 µM β-estradiol for 24 h before sampling. Three independently transformed lines harboring a single copy of each GX-NGG DNA fragment at the PIG1 targeting site were examined. Copy numbers of each <i>NGG</i> and <i>TUA1</i> gene per pg total RNA were estimated by RT-qPCR.</p>", "links"=>[], "tags"=>["transcripts", "gx6-ngg", "gx8-ngg"], "article_id"=>810271, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Amounts_of_NGG_and_TUA1_transcripts_in_GX6_NGG_and_GX8_NGG_lines_/810271", "title"=>"Amounts of <i>NGG</i> and <i>TUA1</i> transcripts in GX6-NGG and GX8-NGG lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218710"], "description"=>"<p>(A, B) Relative transcript levels of <i>NGG</i> in protonemata of GX6-NGG#63 (squares) and GX6-NGG#65 (triangles) line (A) and GX8-NGG#4 (squares) and GX8-NGG#16 (triangles) line (B). Protonemata were immersed in water containing different concentrations of β-estradiol for 24 h before sampling. Relative transcript levels of <i>NGG</i> are normalized to <i>TUA1</i> levels and standardized to a normalized transcript level of protonemata without β-estradiol. Error bars indicate SD of the mean (n = 3).</p>", "links"=>[], "tags"=>["responsiveness", "gx6-ngg", "gx8-ngg"], "article_id"=>810272, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dose_responsiveness_to_946_estradiol_in_the_GX6_NGG_and_GX8_NGG_lines_/810272", "title"=>"Dose responsiveness to β-estradiol in the GX6-NGG and GX8-NGG lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218711"], "description"=>"<p>(A, B) Relative transcript levels of <i>NGG</i> in protonemata of GX6-NGG#63 (A) and GX8-NGG#4 (B) lines. Relative transcript levels of <i>NGG</i> are normalized to <i>TUA1</i> and then standardized to the normalized transcript level of protonemata at 0 h. Protonemata were immersed in water with (+: circles) 1 µM β-estradiol and collected after 0, 1, 3, 6, 12, 24, and 48 h. As a negative control, protonema cultivated without (-: crosses) β-estradiol were collected after 0, 6, 12, and 48 h. Error bars indicate SD of the mean (n = 3).</p>", "links"=>[], "tags"=>["responsiveness", "gx6-ngg", "gx8-ngg"], "article_id"=>810273, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_course_responsiveness_to_946_estradiol_in_the_GX6_NGG_and_GX8_NGG_lines_/810273", "title"=>"Time course responsiveness to β-estradiol in the GX6-NGG and GX8-NGG lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218712"], "description"=>"<p>Fluorescence images of protonemata and a gametophore leaf of the GX6-NGG#63/LGZ2-NmRFP1#11 line. They were immersed in water containing 1 µM β-estradiol for 24 h before microscopy. Fluorescence images of NGG and chlorophyll autofluorescence (top row), NGG (second row), NmRFP1 (third row), and a merged image of NGG and NmRFP1 (bottom row) are indicated. Each picture is at the same magnification. Bar = 200 µm.</p>", "links"=>[], "tags"=>["patterns", "dually", "induced", "ngg"], "article_id"=>810274, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Spatial_expression_patterns_of_dually_induced_NGG_and_NmRFP1_/810274", "title"=>"Spatial expression patterns of dually induced NGG and NmRFP1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218713"], "description"=>"<p>(A, B, C) Transcript amounts of <i>NGG</i> (A), <i>NmRFP1</i> (B), and <i>TUA1</i> (C) in protonemata of GX6-NGG#63/LGZ2-NmRFP lines. Protonemata were immersed in water with (+: circles) or without (-: crosses) 1 µM β-estradiol for 24 h before sampling. Copy numbers of <i>NGG</i>, <i>NmRFP1</i>, and <i>TUA1</i> genes per pg total RNA were estimated by RT-qPCR.</p>", "links"=>[], "tags"=>["amounts", "dually", "induced", "transgenic"], "article_id"=>810275, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.g008"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transcript_amounts_for_dually_induced_NGG_and_NmRFP1_in_transgenic_P_patens_lines_/810275", "title"=>"Transcript amounts for dually induced <i>NGG</i> and <i>NmRFP1</i> in transgenic <i>P</i><i>. patens</i> lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-27 02:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1218728", "https://ndownloader.figshare.com/files/1218730", "https://ndownloader.figshare.com/files/1218731", "https://ndownloader.figshare.com/files/1218733", "https://ndownloader.figshare.com/files/1218734", "https://ndownloader.figshare.com/files/1218735", "https://ndownloader.figshare.com/files/1218736", "https://ndownloader.figshare.com/files/1218738", "https://ndownloader.figshare.com/files/1218739", "https://ndownloader.figshare.com/files/1218740", "https://ndownloader.figshare.com/files/1218741"], "description"=>"<div><p>Inducible transgene expression provides a useful tool to analyze gene function. The moss <i>Physcomitrella</i><i>patens</i> is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The <i>P</i><i>. patens</i> system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in <i>P</i><i>. patens</i><i>.</i></p> </div>", "links"=>[], "tags"=>["moss"], "article_id"=>810280, "categories"=>["Biological Sciences"], "users"=>["Minoru Kubo", "Akihiro Imai", "Tomoaki Nishiyama", "Masaki Ishikawa", "Yoshikatsu Sato", "Tetsuya Kurata", "Yuji Hiwatashi", "Ralf Reski", "Mitsuyasu Hasebe"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0077356.s001", "https://dx.doi.org/10.1371/journal.pone.0077356.s002", "https://dx.doi.org/10.1371/journal.pone.0077356.s003", "https://dx.doi.org/10.1371/journal.pone.0077356.s004", "https://dx.doi.org/10.1371/journal.pone.0077356.s005", "https://dx.doi.org/10.1371/journal.pone.0077356.s006", "https://dx.doi.org/10.1371/journal.pone.0077356.s007", "https://dx.doi.org/10.1371/journal.pone.0077356.s008", "https://dx.doi.org/10.1371/journal.pone.0077356.s009", "https://dx.doi.org/10.1371/journal.pone.0077356.s010", "https://dx.doi.org/10.1371/journal.pone.0077356.s011"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/System_for_Stable_Estradiol_Inducible_Gene_Expression_in_the_Moss_Physcomitrella_patens_/810280", "title"=>"System for Stable β-Estradiol-Inducible Gene Expression in the Moss <i>Physcomitrella</i><i> patens</i>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-09-27 02:20:22"}

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Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Cell biology", "average_usage"=>[272, 472, 600, 713, 815, 911, 1004, 1094, 1185, 1273, 1358, 1441]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[281, 484, 611, 728, 835, 934, 1030, 1123, 1214, 1299, 1383, 1464]}]}
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