2000 Fiscal Year Annual Research Report

マキシザイムを利用したホヤ胚遺伝子機能抑制実験系の確立

Research Project

Project/Area Number	11680728
Research Institution	Konan University
Principal Investigator	西方敬人甲南大学, 理学部, 助教授 (80212116)
Keywords	DNAザイム / ホヤ / RT-PCR / メトキシ化 / 母性mRNA / アンチセンス・テクノロジー / 機能破壊
Research Abstract	ホヤは卵内にあらかじめ母性mRNAが局在し、それらの働きが体を作り上げていくうえで非常に重要であると考えられている。しかし、特定の遺伝子の機能破壊実験系が確立されておらず、遺伝子の機能の証明が大変難しかった。そこで、近年in vivoへの応用が様々な分野で注目されている機能核酸を用いて、母性mRNAの機能阻害実験系を確立することを目的とした。昨年度、ヒトpol IIIプロモーター配列がホヤ胚で有効に機能出来ないことが明らかになったことで、マキシザイムの開発は一旦休止することとなった。そのかわり、細胞内でより安定なDNAザイムを用いた実験を中心に行った。標的遺伝子の適当な配列を選び、切断活性のあるDZ、およびポイントミューテーションを入れることで切断活性を無くしたIDZを設計し、それらを未受精卵に注入した。様々な濃度で注入し、1-12時間放置して卵内の標的mRNAが消滅するかどうかRT-PCRを用いて調べ、最適条件を検討した。いくつかのDZでは、0.125mg/mlの濃度で注入し、6時間放置することで、標的RNAを消失させることができた。ところがあるIDZには、標的遺伝子を消失させる効果があることが分かった。勿論、in vitroにおいてこのIDZに切断活性は見られない。この設計ではアンチセンス効果が現れたものだと考えられ、RNaseが豊富に存在する卵内で用いるためのDNAザイム設計の注意点とし浮かび上がった。一方、標的遺伝子を消失させた卵を発生させてその効果を調べたところ、DZ、IDZともに同様の発生異常を示し、非特異的毒性が予想以上に高いことが明らかになった。特異的効果を得るための条件設定は、非常に微妙なものになると考えられた。今後さらに多くのDZおよびIDZを設計し、母性mRNAを特異的かつ効率的に破壊するDNAザイムの設計法、ならびに実験条件をより明確にする予定である。

Research Products
(15 results)

All Other

All Publications (15 results)

[Publications] Nishikata,T.: "RNA-binding proteins in ascidian development.""Biology of Ascidian" Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)
[Publications] Tanaka,K.J.: "Functional analysis of Ciona intestinalis Y-box protein.""Biology of Ascidian" Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)
[Publications] Makabe,K.W.: "Maternal genetic information stored in fertilized eggs of the ascidian, Halocynthia roretzi.""Biology of Ascidian"Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)
[Publications] Hatano,K.: "Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)."Phycological Research. 48. 143-148 (2000)
[Publications] Matsugu,H.: "Improved antigen detection on Western-blots."Technical Tips OnLine. T02043 (2000)
[Publications] Tanaka,K.J.: "The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell stage embryo."Development, Genes and Evolution. 210. 464-466 (2000)
[Publications] Tanaka,K.J.: "An ascidian glycine-rich RNA-binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis."Gene. 243. 207-214 (2000)
[Publications] Kawashima,T.: "Expression patterns of musashi homologus of the ascidians, Halocynthia roretzi and Ciona intestinalis."Development, Genes and Evolution. 210. 162-165 (2000)
[Publications] Tanaka,K.J.: "A non-radioactive gel shift protocol enables recovery of RNA-binding proteins."Technical Tips OnLine. T01794 (1999)
[Publications] Nakamori,R.: "Characterization of MTR1 : A novel protein correlated with the myoplasmic movement during ascidian ooplasmic segregation."Memoirs of Konan University, Science Series. 46. 25-33 (1999)
[Publications] Okubo,M.-A.: "Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells."Development, Growth & Differentiation. 41. 381-389 (1999)
[Publications] Nishikata,T.: "The CAB (Centrosome-Attracting Body), microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo."Developmental Biology. 209. 72-85 (1999)
[Publications] Chiba,S.: "Interactions between cytoskeletal components during ooplasmic segregation in the myoplasm of ascidian eggs."Development, Growth & Differentiation. 41. 265-272 (1999)
[Publications] Wada,M.R.: "The expression of the protochordate homologue of the proteasome regulatory subunit Rpn 12 is transcriptionally and post-translationally regulated during cleavage stage."Zoological Science. 16. 125-129 (1999)
[Publications] Nishida,H.: "Maternal cytoplasmic factors for generation of unique cleavage patterns in animal embryos."Current Topics in Developmental Biology. 34. 1-37 (1999)

2000 Fiscal Year Annual Research Report

マキシザイムを利用したホヤ胚遺伝子機能抑制実験系の確立

Principal Investigator

西方 敬人 甲南大学, 理学部, 助教授 (80212116)

Research Products

[Publications] Nishikata,T.: "RNA-binding proteins in ascidian development.""Biology of Ascidian" Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)

[Publications] Tanaka,K.J.: "Functional analysis of Ciona intestinalis Y-box protein.""Biology of Ascidian" Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)

[Publications] Makabe,K.W.: "Maternal genetic information stored in fertilized eggs of the ascidian, Halocynthia roretzi.""Biology of Ascidian"Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)

[Publications] Hatano,K.: "Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)."Phycological Research. 48. 143-148 (2000)

[Publications] Matsugu,H.: "Improved antigen detection on Western-blots."Technical Tips OnLine. T02043 (2000)

[Publications] Tanaka,K.J.: "The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell stage embryo."Development, Genes and Evolution. 210. 464-466 (2000)

[Publications] Tanaka,K.J.: "An ascidian glycine-rich RNA-binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis."Gene. 243. 207-214 (2000)

[Publications] Kawashima,T.: "Expression patterns of musashi homologus of the ascidians, Halocynthia roretzi and Ciona intestinalis."Development, Genes and Evolution. 210. 162-165 (2000)

[Publications] Tanaka,K.J.: "A non-radioactive gel shift protocol enables recovery of RNA-binding proteins."Technical Tips OnLine. T01794 (1999)

[Publications] Nakamori,R.: "Characterization of MTR1 : A novel protein correlated with the myoplasmic movement during ascidian ooplasmic segregation."Memoirs of Konan University, Science Series. 46. 25-33 (1999)

[Publications] Okubo,M.-A.: "Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells."Development, Growth & Differentiation. 41. 381-389 (1999)

[Publications] Nishikata,T.: "The CAB (Centrosome-Attracting Body), microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo."Developmental Biology. 209. 72-85 (1999)

[Publications] Chiba,S.: "Interactions between cytoskeletal components during ooplasmic segregation in the myoplasm of ascidian eggs."Development, Growth & Differentiation. 41. 265-272 (1999)

[Publications] Wada,M.R.: "The expression of the protochordate homologue of the proteasome regulatory subunit Rpn 12 is transcriptionally and post-translationally regulated during cleavage stage."Zoological Science. 16. 125-129 (1999)

[Publications] Nishida,H.: "Maternal cytoplasmic factors for generation of unique cleavage patterns in animal embryos."Current Topics in Developmental Biology. 34. 1-37 (1999)

西方敬人甲南大学, 理学部, 助教授 (80212116)