Protein production using Daphnia magna

• Project/Area Number: 15K14897
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Insect science
• Research Institution: Osaka University
• Principal Investigator: Watanabe Hajime 大阪大学, 工学研究科, 教授 (80212322)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2015: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: ミジンコ / タンパク質産生 / ゲノム編集 / ビテロジェニン / 細胞間輸送 / シグナルペプチド

Outline of Final Research Achievements

We aimed to produce artificial protein in Daphnia magna using genome editing technique. Firstly we made a synthetic gene that has　a signal peptide necessary for secretion with green fluorescent protein (GFP) and confirmed that the product of the synthetic gene was secreted into hemolymph. Secondly we fused a gene fragment responsible for incorporation into eggs with the synthetic gene. This gene product was successfully synthesized in somatic cells ant transported to eggs.

Research Products

• [Journal Article] Construction of an in Vitro Gene Screening System of the E. coli EmrE Transporter Using Liposome Display. (2016)
  • Author(s): Uyeda, A., Nakayama, S., Kato, Y., Watanabe, H. and Matsuura, T.
  • Journal Title: Anal. Chem. Volume: 88 Issue: 24 Pages: 12028-12035
  • DOI: 10.1021/acs.analchem.6b02308
  • Peer Reviewed
• [Journal Article] In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes (2016)
  • Author(s): Ohta, N., Kato, Y., Watanabe, H., Mori, H., and *Matsuura, T
  • Journal Title: Sci Rep Volume: 6 Issue: 1 Pages: 36252-36252
  • DOI: 10.1038/srep36466
  • Peer Reviewed / Open Access
• [Journal Article] TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna. (2016)
  • Author(s): Nakanishi, T., Kato, Y., Matsuura, T., Watanabe, H.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 3652-3652
  • DOI: 10.1038/srep36252
  • Peer Reviewed / Open Access
• [Journal Article] Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna. (2016)
  • Author(s): Mohamad Ishak, N. S., Kato, Y., Matsuura, T., Watanabe, H.
  • Journal Title: PLoS One Volume: 11 Issue: 5 Pages: e0154636-e0154636
  • DOI: 10.1371/journal.pone.0154636
  • Peer Reviewed / Open Access
• [Journal Article] Betaproteobacteria Limnohabitans strains increase fecundity in the crustacean Daphnia magna: symbiotic relationship between major bacterioplankton and zooplankton in freshwater ecosystem. (2016)
  • Author(s): Peerakietkhajorn S, Kato Y, Kasalicky V, Matsuura T, Watanabe H
  • Journal Title: Environ Microbiol. Volume: in press Issue: 8 Pages: 2366-2374
  • DOI: 10.1111/1462-2920.12919
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Growth evaluation method by live imaging of Daphnia magna and its application to the estimation of an insect growth regulator. (2015)
  • Author(s): Suzuki A, Kato Y, Matsuura T, Watanabe H
  • Journal Title: J Appl Toxicol Volume: 35 Pages: 68-74
  • Peer Reviewed
• [Journal Article] Symbiotic bacteria contribute to increasing the population size of a freshwater crustacean, Daphnia magna. (2015)
  • Author(s): Peerakietkhajorn S, Tsukada K, Kato Y, Matsuura T, Watanabe H
  • Journal Title: Environ Microbiol Rep Volume: 7 Issue: 2 Pages: 364-372
  • DOI: 10.1111/1758-2229.12260
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] TALEN-mediated homologous recombination in Daphnia magna. (2015)
  • Author(s): Nakanishi T, Kato Y, Matsuura T, Watanabe H
  • Journal Title: Sci Rep Volume: 5 Issue: 1 Pages: 18312-18312
  • DOI: 10.1038/srep18312
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna (2015)
  • Author(s): 内藤彰子、加藤泰彦、中西貴士、松浦友亮、渡邉肇
  • Journal Title: Biology OPEN Volume: 13 Issue: 3 Pages: 364-369
  • DOI: 10.1242/bio.20149738
  • Peer Reviewed / Open Access
• [Journal Article] Liposome-based in vitro evolution of aminoacyl-tRNA synthetase for enhanced pyrrolysine derivative incorporation. (2015)
  • Author(s): Uyeda A, Watanabe T, Kato Y, Watanabe H, Yomo T, Hohsaka T, Matsuura T
  • Journal Title: Chembiochem Volume: 16 Issue: 12 Pages: 1797-1802
  • DOI: 10.1002/cbic.201500174
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] ミジンコの性決定遺伝子を活性化する長鎖ノンコーディングRNAの機能解析 (2016)
  • Author(s): 加藤泰彦, Nong Dang Quang, 渡邉肇（優秀ポスター賞を受賞）
  • Organizer: 第３９回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2016-11-29
• [Presentation] The bZIP transcription factor Vrille up-regulates the environmental sex-determining gene Doublesex1 in Daphnia magna (2016)
  • Author(s): Nur Syafiqah Mohamad Ishak、Quang D. Nong、Yasuhiko Kato、渡邉 肇
  • Organizer: 第３９回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2016-11-29
• [Presentation] TALEN-mediated knock-in of a reporter gene for visualizing expression of the sex-determining gene dsx1 in Daphnia magna (2016)
  • Author(s): Nur Syafiqah Mohamad Ishak、Quang D. Nong、Yasuhiko Kato、渡邉 肇
  • Organizer: 第３９回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2016-11-29
• [Presentation] Male production mechanism in parthenogenetic crustacean, Daphnia Magna (2016)
  • Author(s): Hajime Watanabe, Quan D. Nong, Nur Syafiqah Mohamad Ishak, Yasuhiko Kato
  • Organizer: The 22nd International Congress of Zoology
  • Place of Presentation: 沖縄科学技術大学院大学
  • Year and Date: 2016-11-14
  • Int'l Joint Research / Invited
• [Presentation] Longevity lesson from freshwater crustaceans (2016)
  • Author(s): Hajime Watanabe
  • Organizer: The 5th Beneficial Microbes Conference
  • Place of Presentation: Hotel Casa 400 オランダ
  • Year and Date: 2016-10-08
  • Int'l Joint Research / Invited
• [Presentation] オオミジンコにおける組換えタンパク質の卵への輸送系の確立 (2016)
  • Author(s): 辻勇祐、加藤泰彦、渡邉肇
  • Organizer: 第68回日本生物工学会
  • Place of Presentation: 富山国際会議場
  • Year and Date: 2016-09-28
• [Presentation] ミジンコにおける2Aペプチドを用いたバイシストロニック発現システムの構築 (2016)
  • Author(s): 熊谷 仁志, 加藤 泰彦, 松浦 友亮, 渡邉 肇
  • Organizer: 第68回日本生物工学会
  • Place of Presentation: 富山国際会議場
  • Year and Date: 2016-09-28
• [Presentation] A long noncoding RNA regulates an environmental sex-determining gene doublesex1 in Daphnia (2016)
  • Author(s): 加藤泰彦 渡邉肇
  • Organizer: RNA2016
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 2016-06-29
  • Int'l Joint Research
• [Presentation] 「環境指標生物の毒性影響可視化のための遺伝子工学的手法の開発」 (2016)
  • Author(s): 「環境指標生物の毒性影響可視化のための遺伝子工学的手法の開発」
  • Organizer: 第43回日本毒性学会学術年会
  • Place of Presentation: ウインクあいち
  • Year and Date: 2016-06-28
• [Presentation] Development of transgenic Daphnia (2015)
  • Author(s): Yasuhiko KATO, Takashi NAKANISHI, Tomoaki MATSUURA, Hajime WATANABE
  • Organizer: Pacifichem 2015
  • Place of Presentation: ハワイ、USA
  • Year and Date: 2015-12-15
  • Int'l Joint Research
• [Presentation] Analysis of Transcription Activation of the Environmental Sex Determining Gene Doublesex1 in Daphnia magna (2015)
  • Author(s): Syafiqah Ishak, Yasuhiko Kato, Tomoaki Matsuura, Hajime Watanabe
  • Organizer: 第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会
  • Place of Presentation: 神戸国際会議場（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] An lncRNA regulates an environmental sex determination in Daphnia magna (2015)
  • Author(s): Yasuhiko Kato, Hajime Watanabe
  • Organizer: 第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会
  • Place of Presentation: 神戸国際会議場（兵庫県・神戸市）
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] Eye Color Gene Scarlet as a Visible Marker for Daphnia magna Transgenesis (2015)
  • Author(s): Nur Izzatur Binti Ismail, Yasuhiko Kato, Tomoaki Matsuura, Hajime Watanabe
  • Organizer: 第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会
  • Place of Presentation: 神戸国際会議場（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] Use of the bicistronic expression involving viral T2A peptide in Daphnia magna (2015)
  • Author(s): Hitoshi Kumagai, Yasuhiko Kato, Tomoaki Matsuura, Hajime Watanabe
  • Organizer: 第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会
  • Place of Presentation: 神戸国際会議場（兵庫県・神戸市）
  • Year and Date: 2015-12-01
• [Presentation] Efficient DNA integration into germ line genome via TALEN in Daphnia magna (2015)
  • Author(s): Takashi Nakanishi, Yasuhiko Kato, Tomoaki Matsuura, Hajime Watanabe
  • Organizer: Conference on Transposition and Genome Engineering 2015
  • Place of Presentation: 奈良春日野国際フォーラム(奈良県･奈良市）
  • Year and Date: 2015-11-17
  • Int'l Joint Research
• [Presentation] ミジンコのゲノム操作による環境性決定メカニズムの解明 (2015)
  • Author(s): 加藤泰彦、中西貴士、渡邉肇
  • Organizer: 「細胞を創る」研究会8.0
  • Place of Presentation: 大阪大学（大阪府・吹田市）
  • Year and Date: 2015-11-12
  • Invited
• [Presentation] A long noncoding RNA regulates a sex-determining gene doublesex1 in the crustacean Daphnia magna (2015)
  • Author(s): Yasuhiko Kato, Hajime Watanabe
  • Organizer: EMBL Symposia "Noncoding Genome"
  • Place of Presentation: ハイデルベルグ、ドイツ
  • Year and Date: 2015-10-18
  • Int'l Joint Research
• [Presentation] Efficient DNA integration into germ line genome via programmable nucleases in Daphnia magna (2015)
  • Author(s): Takashi Nakanishi, Yasuhiko Kato, Tomoaki Matsuura, Hajime Watanabe
  • Organizer: Cold Spring Harbor Laboratory Meetings & Courses Program "GENOME ENGINEERING: THE CRISPR/CAS REVOLUTION"
  • Place of Presentation: コールドスプリングハーバー、米国
  • Year and Date: 2015-09-24
  • Int'l Joint Research
• [Presentation] ミジンコの生殖戦略 単為生殖から有性生殖への転換点の解析 (2015)
  • Author(s): 渡邉肇
  • Organizer: 日本動物学会第86回大会
  • Place of Presentation: 朱鷺メッセ（新潟県・新潟市）
  • Year and Date: 2015-09-17
  • Invited
• [Presentation] 環境指標生物オオミジンコにおけるTALENを用いた外来遺伝子導入技術の開発 (2015)
  • Author(s): 中西貴士、加藤泰彦、松浦友亮、渡邉肇
  • Organizer: 生物工学若手研究者の集い夏のセミナー2015
  • Place of Presentation: 名古屋大学（愛知県・名古屋市）
  • Year and Date: 2015-07-11
• [Presentation] オオミジンコを用いた毒性試験における共生菌の影響 (2015)
  • Author(s): Patcharaporn Manakul, Peerakietkhajorn Saranya, 松浦 友亮、加藤 泰彦、渡邉 肇
  • Organizer: 第42回日本毒性学会学術年会
  • Place of Presentation: 金沢大学(石川県・金沢市）
  • Year and Date: 2015-06-29
• [Presentation] ホルモン活性検出用 のトランスジェニックミジンコの創出 (2015)
  • Author(s): 中西 貴士，浅田 実希，加藤 康彦，渡邉肇
  • Organizer: 第24回環境化学討論会
  • Place of Presentation: 札幌コンベンションセンター(北海道・札幌市）
  • Year and Date: 2015-06-24
• [Presentation] Development of transgenic Daphnia for monitoring hormonal activities of chemicals (2015)
  • Author(s): Takashi NAKANISHI, Miki ASADA, Yasuhiko KATO, Hajime WATANABE
  • Organizer: SETAC Europe 25th Annual Meeting
  • Place of Presentation: バルセロナ、スペイン
  • Year and Date: 2015-05-03
  • Int'l Joint Research
• [Remarks] 大阪大学大学院工学研究科生命先端工学専攻渡邉研究室
  • URL: http://www.bio.eng.osaka-u.ac.jp/ez/
• [Remarks] 大阪大学工学研究科生命先端工学専攻渡邉研究室研究内容
  • URL: http://www.bio.eng.osaka-u.ac.jp/ez/project/index.html