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In vivo gene function analysis using lentiviral vector

Research Project

Project/Area Number 20670006
Research Category

Grant-in-Aid for Young Scientists (S)

Allocation TypeSingle-year Grants
Research Field Laboratory animal science
Research InstitutionOsaka University

Principal Investigator

IKAWA Masahito  大阪大学, 微生物病研究所, 教授 (20304066)

Project Period (FY) 2008 – 2012
Project Status Completed (Fiscal Year 2012)
Budget Amount *help
¥96,850,000 (Direct Cost: ¥74,500,000、Indirect Cost: ¥22,350,000)
Fiscal Year 2012: ¥20,800,000 (Direct Cost: ¥16,000,000、Indirect Cost: ¥4,800,000)
Fiscal Year 2011: ¥20,800,000 (Direct Cost: ¥16,000,000、Indirect Cost: ¥4,800,000)
Fiscal Year 2010: ¥20,800,000 (Direct Cost: ¥16,000,000、Indirect Cost: ¥4,800,000)
Fiscal Year 2009: ¥20,800,000 (Direct Cost: ¥16,000,000、Indirect Cost: ¥4,800,000)
Fiscal Year 2008: ¥13,650,000 (Direct Cost: ¥10,500,000、Indirect Cost: ¥3,150,000)
Keywordsレンチウイルス / ジーントラップ / バイオリソース / 生殖 / 実験動物 / 不妊 / ノックアウト / 発生工学 / 疾患モデル / 胎盤 / 妊娠高血圧症候群
Research Abstract

In the present study, we developed the lentiviral vector system for random mutagenesis in mice. Next we analyzed reproductive failures found in the mice we created. Consequently, we found that genes coding testis specific proteins, Calr3, Pdilt, Tex101,and Pmis2are required for the maturation of sperm fertilizing protein, ADAM3, and male fertility. We further developed the preeclampsia model mice by placenta specific gene manipulation system.

Assessment Rating
Verification Result (Rating)

A

Report

(7 results)
  • 2013 Research Progress Assessment (Verification Result) ( PDF )
  • 2012 Annual Research Report   Final Research Report ( PDF )
  • 2011 Annual Research Report
  • 2010 Annual Research Report   Self-evaluation Report ( PDF )
  • 2008 Annual Research Report
  • Research Products

    (63 results)

All 2013 2012 2011 2010 2009 2008 Other

All Journal Article (35 results) (of which Peer Reviewed: 35 results) Presentation (20 results) Book (1 results) Remarks (5 results) Patent(Industrial Property Rights) (2 results)

  • [Journal Article] Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa2013

    • Author(s)
      FujiharaY, Tokuhiro K, Muro Y, Kondoh G, Araki Y, Ikawa M, Okabe M
    • Journal Title

      PNAS

      Volume: 110(20) Issue: 20 Pages: 8111-6

    • DOI

      10.1073/pnas.1222166110

    • Related Report
      2012 Annual Research Report 2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration2013

    • Author(s)
      Nojima S, et al.
    • Journal Title

      Nat Commun

      Volume: 4 Issue: 1 Pages: 1406-1406

    • DOI

      10.1038/ncomms2420

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article]2012

    • Author(s)
      Yamaguchi R, Fujihara Y, Ikawa M, Okabe M.Tokuhiro K, Ikawa M, Benham AM, Okabe M
    • Journal Title

      Proc Natl Acad Sci U S A

      Volume: 109 Issue: 10 Pages: 3850-5

    • DOI

      10.1073/pnas.1117963109

    • Related Report
      2012 Final Research Report 2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] SPACA1-deficient male mice are infertile with abnormally shaped sperm heads reminiscent of globozoospermia2012

    • Author(s)
      Fujihara Y, Satouh Y, Inoue N, Isotani A, Ikawa M, Okabe M
    • Journal Title

      Development

      Volume: 139 Issue: 19 Pages: 3583-9

    • DOI

      10.1242/dev.081778

    • NAID

      120006957401

    • Related Report
      2012 Annual Research Report 2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO12012

    • Author(s)
      Satouh Y, Inoue N, Ikawa M, Okabe M
    • Journal Title

      Journal of Cell Science

      Volume: 125 Pages: 4985-4990

    • DOI

      10.1242/jcs.100867

    • NAID

      120006957400

    • Related Report
      2012 Annual Research Report 2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa2012

    • Journal Title

      Mol Biol Cell

      Volume: 23 Issue: 14 Pages: 2671-9

    • DOI

      10.1091/mbc.e11-12-1025

    • Related Report
      2012 Annual Research Report 2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Neutrophil infiltration during inflammation is regulated by PILRalpha via modulation of integrin activation2012

    • Author(s)
      Wang J, et al.
    • Journal Title

      Nat Immunol

      Volume: 14 Issue: 1 Pages: 2019-2025

    • DOI

      10.1038/ni.2456

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Production of mouse pups from germline transmission-failed knockout chimeras.2012

    • Author(s)
      Fujihara Y., et al.
    • Journal Title

      Transgenic Res

      Volume: 22 Issue: 1 Pages: 195-200

    • DOI

      10.1007/s11248-012-9635-x

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] GPHR-dependent functions of the Golgi apparatus are essential for the formation of lamellar granules and the skin2012

    • Author(s)
      Tarutani M, et al.
    • Journal Title

      J Invest Dermatol

      Volume: 132 Issue: 8 Pages: 2019-2025

    • DOI

      10.1038/jid.2012.100

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cold-inducible RNA-binding protein (Cirp) interacts with Dyrk1b/Mirk and promotes proliferation of immature male germ cells in mice2012

    • Author(s)
      Masuda T, et al.
    • Journal Title

      Proc Natl Acad Sci USA

      Volume: 109 Issue: 27 Pages: 10885-108901

    • DOI

      10.1073/pnas.1121524109

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Tetraspanin-interacting protein IGSF8 is dispensable for mouse fertility.2012

    • Author(s)
      Inoue N., et al.
    • Journal Title

      Fertil Steril

      Volume: 98 Issue: 2 Pages: 465-470

    • DOI

      10.1016/j.fertnstert.2012.04.029

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Mice Deficient in Ficolin, a Lectin Complement Pathway Recognition Molecule, Are Susceptible to Streptococcus pneumoniae Infection2012

    • Author(s)
      Endo Y, et al.
    • Journal Title

      J Immunol

      Volume: 189 Issue: 12 Pages: 5860-5866

    • DOI

      10.4049/jimmunol.1200836

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] AMPD3-deficient mice exhibit increased erythrocyte ATP levels but anemia not improved due to PK deficiency2012

    • Author(s)
      Cheng J, et al.
    • Journal Title

      Genes Cells

      Volume: 17 Issue: 11 Pages: 913-922

    • DOI

      10.1111/gtc.12006

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Characterization of inclusion bodies with cytoprotective properties formed by seipinopathy-linked mutant seipin2012

    • Author(s)
      Ito D, Yagi T, Ikawa M, Suzuki N
    • Journal Title

      Human Molecular Genetics

      Volume: 21 Issue: 3 Pages: 635-46

    • DOI

      10.1093/hmg/ddr497

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model2011

    • Author(s)
      Kumasawa K, Ikawa M, Kidoya H, Hasuwa H, Saito-Fujita T, Morioka Y, Takakura N, Kimura T, Okabe M
    • Journal Title

      Proc Natl Acad Sci U S A

      Volume: 108 Issue: 4 Pages: 1451-5

    • DOI

      10.1073/pnas.1011293108

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility.2011

    • Author(s)
      Ikawa M*#, Tokuhiro K*, Yamaguchi R, Benham AM, Tamura T, Wada I, Satouh Y, Inoue N, Okabe M
    • Journal Title

      J Biol Chem

      Volume: 286 Issue: 7 Pages: 5639-5646

    • DOI

      10.1074/jbc.m110.140152

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs2011

    • Author(s)
      Inoue N, Satouh Y, Ikawa M, Okabe M, Yanagimachi R
    • Journal Title

      Proc Natl Acad Sci U S A

      Volume: 108 Issue: 50 Pages: 20008-20011

    • DOI

      10.1073/pnas.1116965108

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes2011

    • Author(s)
      Mitsutake S., et al
    • Journal Title

      J Biol Chem

      Volume: 286 Issue: 32 Pages: 28544-55

    • DOI

      10.1074/jbc.m111.255646

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Homologous recombination in rat germline stem cells2011

    • Author(s)
      Kanatsu-Shinohara M., et al
    • Journal Title

      Biol Reprod

      Volume: 85 Issue: 1 Pages: 208-17

    • DOI

      10.1095/biolreprod.111.090837

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Formation of a thymus from rat ES cells in xenogeneic nude mouse<--> rat ES chimeras2011

    • Author(s)
      Isotani A., et al
    • Journal Title

      Genes Cells

      Volume: 16 Issue: 4 Pages: 397-405

    • DOI

      10.1111/j.1365-2443.2011.01495.x

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] From the Cover : Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model.2011

    • Author(s)
      Kumasawa K*, Ikawa M*#, Kidoya H, Hasuwa H, Saito-Fujita T, Morioka Y, Takakura N, Kimura T, Okabe M.
    • Journal Title

      Proc Natl Acad Sci USA 108

      Pages: 1451-1455

    • Related Report
      2010 Self-evaluation Report
    • Peer Reviewed
  • [Journal Article] Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility.2011

    • Author(s)
      Ikawa M*#, Tokuhiro K*, Yamaguchi R, Benham AM, Tamura T, Wada I, Satouh Y, Inoue N, Okabe M.
    • Journal Title

      J Biol Chem 286

      Pages: 5639-5646

    • Related Report
      2010 Self-evaluation Report
    • Peer Reviewed
  • [Journal Article] Fertilization: a sperm's journey to and interaction with the oocyte.2010

    • Author(s)
      Ikawa M, Inoue N, Benham AM, Okabe M#.
    • Journal Title

      J Clin Invest

      Volume: 120 Issue: 4 Pages: 984-994

    • DOI

      10.1172/jci41585

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Fertilization : a sperm's journey to and interaction with the oocyte.2010

    • Author(s)
      Ikawa M, Inoue N, Benham AM, Okabe M#.
    • Journal Title

      J Clin Invest 120

      Pages: 984-994

    • Related Report
      2010 Self-evaluation Report
    • Peer Reviewed
  • [Journal Article] Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model2010

    • Author(s)
      Kumasawa K, et.al.
    • Journal Title

      Proc Natl Acad Sci U S A

      Volume: 108(4) Pages: 1451-5

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility2010

    • Author(s)
      Ikawa M, et.al.
    • Journal Title

      J Biol Chem

      Volume: 286(7) Pages: 5639-46

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Fertilization : a sperm's journey to and interaction with the oocyte2010

    • Author(s)
      Ikawa M, et.al.
    • Journal Title

      J Clin Invest

      Volume: 120(4) Pages: 984-94

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Placenta-specific gene activation and inactivation using integrase-defective lentiviral vectors with the Cre/LoxP system2009

    • Author(s)
      Morioka Y, Isotani A, Oshima RG, Okabe M, Ikawa M#
    • Journal Title

      Genesis

      Volume: 47 Issue: 12 Pages: 793-798

    • DOI

      10.1002/dvg.20563

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors.2009

    • Author(s)
      Okada Y, Ueshin Y, Hasuwa H, Takumi K, Okabe M, Ikawa M#
    • Journal Title

      Genesis

      Volume: 47 Issue: 4 Pages: 217-223

    • DOI

      10.1002/dvg.20469

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Placenta-specific gene activation and inactivation using integrase-defective lentiviral vectors with the Cre/LoxP system2009

    • Author(s)
      Morioka Y, Isotani A, Oshima RG, Okabe M, Ikawa M#.
    • Journal Title

      Genesis 47

      Pages: 793-798

    • Related Report
      2010 Self-evaluation Report
    • Peer Reviewed
  • [Journal Article] Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors.2009

    • Author(s)
      Okada Y, Ueshin Y, Hasuwa H, Takumi K, Okabe M, Ikawa M#.
    • Journal Title

      Genesis 47

      Pages: 217-223

    • Related Report
      2010 Self-evaluation Report
    • Peer Reviewed
  • [Journal Article] Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors2009

    • Author(s)
      Okada, Y. et. al.
    • Journal Title

      Genesis (in press)

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Putative sperm fusion protein IZUMO and the role of N-glycosylation.2008

    • Author(s)
      Inoue, N. et. al.
    • Journal Title

      Biochem Biophys Res Commun 377

      Pages: 910-4

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cd52, known as a major maturation-associated sperm membrane antigen secreted from the epididymis, is not required for fertilization in the mouse.2008

    • Author(s)
      Yamaguchi, R. et. al.
    • Journal Title

      Genes Cells 13

      Pages: 335-45

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Mechanisms of sperm-egg interactions emerging from gene-manipulated animals.2008

    • Author(s)
      Ikawa, M. et. al.
    • Journal Title

      Int J Dev Biol 52

      Pages: 657-64

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Presentation] ER chaperones, ADAM3, and sperm fertilizing ability2013

    • Author(s)
      伊川正人
    • Organizer
      日本発生生物学会第46回大会
    • Place of Presentation
      松江
    • Year and Date
      2013-05-31
    • Related Report
      2012 Final Research Report
  • [Presentation] 遺伝子改変動物を用いた生殖メカニズムの解明2013

    • Author(s)
      伊川正人
    • Organizer
      第1回実験動物科学シンポジウム
    • Place of Presentation
      京都大学 百周年時計台記念館(招待講演)
    • Year and Date
      2013-02-01
    • Related Report
      2012 Annual Research Report
  • [Presentation] 小胞体シャペロンと精子受精能力2012

    • Author(s)
      伊川正人
    • Organizer
      関西生殖発生毒性フォーラム
    • Place of Presentation
      大阪市立弁天町市民学習センター(招待講演)
    • Year and Date
      2012-11-10
    • Related Report
      2012 Annual Research Report
  • [Presentation] Placental Gene Manipulation and Its Application in the Study of Placental Biology and Diseases2012

    • Author(s)
      Masahito Ikawa
    • Place of Presentation
      北京, China
    • Year and Date
      2012-05-10
    • Related Report
      2012 Final Research Report
  • [Presentation] Placental Gene Manipulation and Its Application in the Study of Placental Biology and Diseases2012

    • Author(s)
      Masahito Ikawa
    • Organizer
      The 2nd SKLRB Symposia on Frontiers in Reproductive Biology
    • Place of Presentation
      Beijing Xijiao Hotel 中国(招待講演)
    • Year and Date
      2012-05-10
    • Related Report
      2012 Annual Research Report
  • [Presentation] ANZPLA meeting (satellite meeting of WCRB)2011

    • Author(s)
      Masahito Ikawa
    • Organizer
      Lentiviral transduction & placenta-specific gene manipulation to study PE in a mouse model
    • Place of Presentation
      Cairns, Australia
    • Year and Date
      2011-10-07
    • Related Report
      2012 Final Research Report
  • [Presentation] Lentiviral transduction & placenta-specific gene manipulation to study PE in a mouse model2011

    • Author(s)
      Masahito Ikawa
    • Organizer
      World Congress on Reproductive Biology
    • Place of Presentation
      Cairns, Australia(招待講演)
    • Year and Date
      2011-10-07
    • Related Report
      2011 Annual Research Report
  • [Presentation]2011

    • Author(s)
      Masahito Ikawa
    • Organizer
      9th International Calreticulin Workshop
    • Place of Presentation
      Copenhagen, Denmark
    • Year and Date
      2011-08-29
    • Related Report
      2012 Final Research Report
  • [Presentation] Testis specific calreticulin homologue, calsperin, regulates sperm fertility2011

    • Author(s)
      Masahito Ikawa
    • Organizer
      9th International Calreticulin Workshop
    • Place of Presentation
      Copenhagen, Denmark(招待講演)
    • Year and Date
      2011-08-29
    • Related Report
      2011 Annual Research Report
  • [Presentation] 胎盤の遺伝子操作とその応用2011

    • Author(s)
      伊川正人
    • Organizer
      第58回日本実験動物学会総会
    • Place of Presentation
      船堀
    • Year and Date
      2011-05-25
    • Related Report
      2012 Final Research Report
  • [Presentation] 胎盤の遺伝子操作とその応用2011

    • Author(s)
      伊川正人
    • Organizer
      第58回日本実験動物学会総会
    • Place of Presentation
      船堀(招待講演)
    • Year and Date
      2011-05-25
    • Related Report
      2011 Annual Research Report
  • [Presentation] Lentiviral vector mediated gene transfer and its application in mouse genetics.2010

    • Author(s)
      Masahito Ikawa
    • Organizer
      The 4^<th> AFLAS Congress meeting.
    • Place of Presentation
      Taipei
    • Year and Date
      2010-11-11
    • Related Report
      2010 Self-evaluation Report
  • [Presentation] Lentiviral vector mediated gene transfer and its application in mouse genetics2010

    • Author(s)
      Masahito Ikawa
    • Organizer
      The 4th AFLAS Congress meeting
    • Place of Presentation
      台湾台北市 Taipei International Convention Center(中華民国)(招待講演)
    • Year and Date
      2010-11-11
    • Related Report
      2010 Annual Research Report
  • [Presentation] Lentiviral vector mediated gene transfer and its application in mouse genetics2010

    • Author(s)
      Masahito Ikawa
    • Organizer
      Asian Federation of Laboratory Animal Science Associations Annual Meeting
    • Place of Presentation
      Taipei, 中華民国
    • Year and Date
      2010-11-09
    • Related Report
      2012 Final Research Report
  • [Presentation] Placenta specific gene manipulation using lentiviral vectors2009

    • Author(s)
      Masahito Ikawa
    • Organizer
      4th Asian Mouse Mutagenesis Resource Association meeting
    • Place of Presentation
      Singapore
    • Year and Date
      2009-12-17
    • Related Report
      2012 Final Research Report
  • [Presentation] Placenta specific gene manipulation using lentiviral vectors.2009

    • Author(s)
      Masahito ikawa
    • Organizer
      4^<th> AMMRA meeting
    • Place of Presentation
      Kumamoto
    • Year and Date
      2009-12-17
    • Related Report
      2010 Self-evaluation Report
  • [Presentation] 精巣シャペロンが制御する精子の受精能力2009

    • Author(s)
      伊川正人
    • Organizer
      第80回日本動物学会
    • Place of Presentation
      静岡
    • Year and Date
      2009-09-17
    • Related Report
      2012 Final Research Report
  • [Presentation] レンチウイルスベクターを用いた新しいジーントラップ法の開発2009

    • Author(s)
      伊川正人
    • Organizer
      第56回日本実験動物学会総会
    • Place of Presentation
      大宮
    • Year and Date
      2009-05-15
    • Related Report
      2012 Final Research Report
  • [Presentation] ER Chaperones and Sperm Fertility.2009

    • Author(s)
      Masahito Ikawa
    • Organizer
      Keystone Symposia (Frontiers in Reproductive Biology and Regulation of Fertility)
    • Place of Presentation
      Santa Fe
    • Year and Date
      2009-02-01
    • Related Report
      2010 Self-evaluation Report
  • [Presentation] ウイルスベクターを用いた胎盤特異的な遺伝子機能解析2008

    • Author(s)
      伊川正人
    • Organizer
      日本生殖医学会
    • Place of Presentation
      神戸
    • Year and Date
      2008-10-23
    • Related Report
      2012 Final Research Report
  • [Book] 発明者および扶桑薬品2010

    • Author(s)
      妊娠高血圧症候群モデル動物とその治療方法
    • Total Pages
      20100304
    • Publisher
      岡部勝、伊川正人、木村正、熊澤恵一
    • Related Report
      2010 Self-evaluation Report
  • [Remarks]

    • URL

      http://www.egr.biken.osaka-u.ac.jp/information/

    • Related Report
      2012 Final Research Report
  • [Remarks]

    • URL

      http://www.arcid.biken.osaka-u.ac.jp/index.php

    • Related Report
      2012 Final Research Report
  • [Remarks]

    • URL

      http://www.egr.biken.osaka-u.ac.jp/

    • Related Report
      2012 Annual Research Report
  • [Remarks]

    • URL

      http://www.egr.biken.osaka-u.ac.jp/

    • Related Report
      2011 Annual Research Report
  • [Remarks]

    • URL

      http://kumikae01.gen-info.osaka-u.ac.jp/members/ikawa/index.htm

    • Related Report
      2010 Annual Research Report
  • [Patent(Industrial Property Rights)] 栄養外胚葉細胞特異的遺伝子導入法2012

    • Inventor(s)
      岡部勝、伊川正人
    • Industrial Property Rights Holder
      岡部勝、伊川正人
    • Acquisition Date
      2012-06-22
    • Related Report
      2012 Final Research Report
  • [Patent(Industrial Property Rights)] 妊娠高血圧症候群モデル動物とその治療方法2010

    • Inventor(s)
      伊川正人、熊澤恵一、木村正、岡部勝
    • Industrial Property Rights Holder
      発明者および扶桑薬品工業
    • Industrial Property Number
      2010-048237
    • Filing Date
      2010-03-04
    • Related Report
      2012 Final Research Report

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Published: 2008-04-01   Modified: 2019-07-29  

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