JP4414332B2 - アルツハイマーtauタンパク質を発現するトランスジェニック動物 - Google Patents
アルツハイマーtauタンパク質を発現するトランスジェニック動物 Download PDFInfo
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Description
-該分子はGene-Bankの寄託番号NM 173727で示される4反復配列および3反復配列tauタンパク質をコードする完全長tau cDNA配列の開始コドン下流の少なくとも30ヌクレオチドおよび終止コドン上流の少なくとも30ヌクレオチドがそれぞれトランケートされており、
-最小トランケートtauコアはヌクレオチド744-930(配列番号9;tauタンパク質アイソフォーム43に従って番号付け)によりコードされるタンパク質断片を含み、
-該DNA構築物は動物の脳細胞に発現すると神経原繊維的(NF)病状を生じる活性を有するタンパク質をコードする。
(4反復配列tau(tau 43)由来の誘導体をR4で示し、3反復配列tau (tau 43)由来の誘導体をR3で示す。) ここで用いた番号付けはGoedertら(24)に記載の完全長tau cDNAに由来する。
配列番号1(91-1059、R4)
上記cDNA配列は脳内でトランケートtauタンパク質を発現させるために調節配列と結合している。Thy-1遺伝子の修飾(25)はT細胞中の発現をもたらす調節配列が欠失し、トランケートcDNA配列がSpe-IおよびXho制限部位間に導入されるように行われた。修飾構築物は原核性配列が除去され、マイクロインジェクションにより雄前核中に導入された。あらゆる機能的プロモーターまたはプロモーターエンハンサー複合体を上記トランケートtau cDNAの発現に用いることができよう。
(a) 3または4反復配列taucDNA分子の少なくとも最初の30および最後の30ヌクレオチドにわたる欠失を有する二重トランケートtau分子のコーディング配列を保持する組換え原核性クローニングプラスミドの構築、
(b) トランケートtau分子の各DNA配列およびその組み合わせならびに脳での発現または遍在性発現のための適切なプロモーターを有する真核性発現プラスミドの構築、
(c) 該プラスミドを含む細菌の増殖および該プラスミドの増幅および質の高いその抽出、
(d) 該遺伝子構築物のCOS-7細胞へのトランスフェクション、およびwesternブロット技術を用いるその分析、
(e) すべての原核性配列が排除されるように該遺伝子構築物の単離および生成、およびマイクロインジェクションに適した濃度に緩衝液で希釈 (実施例1参照)、
(f) マイクロインジェクションのための遺伝子構築物の確認。
プライマー対A:
センス:5'-GTGGATCTCAAGCCCTCAAG-3'
アンチセンス:5'-GATCCCCTGATTTTGGAGGT-3'
PCR生成物の予想サイズは235ヌクレオチドである。
プライマー対B:
センス:5'-AAGGTGACCTCCAAGTGTGG-3'
アンチセンス:5'-GGTATGCATGGAGGGAGAAG-3'
PCR生成物の予想サイズは438ヌクレオチドである。
-トランス遺伝子がメンデルの法則に従ってトランスジェニック系統を形成する動物の次世代に伝達される(実施例4参照)、
-遺伝したトランス遺伝子は実施例3、図4に示すようにfounder動物および子孫の脳中にトランケートタンパク質を発現する。
-トランケートtauタンパク質の発現は神経原繊維的病状の形成を促進する(実施例5)、
-PHF1免疫反応性NFTは内因性正常ラットtauがNFTの形成に関与することを示した(実施例5)、
-神経原繊維変化はCNSのニューロン中の細胞内封入体およびフィラメントとして現れ、ヒトAD罹患脳中のNFTとホモローガスである、
-免疫蛍光法は、ヒト脳組織に観察される前臨床アルツハイマー病において見られるものと同様の小ロッド様構造の存在を示した(実施例5参照)。
-ADの最も一般的な危険因子である高血圧、
-食習慣により誘発されうる糖尿病(糖尿病はADの重要な危険因子である。)、
-食習慣により誘導されうる高コレステロール血症(ADの別の重要な危険因子)。
別の好ましい態様において、本発明は
−該動物の神経原繊維病状の変化を検出し、該動物の神経行動学的変化を測定し、該動物の認知スコアを測定し、動物組織および体液中のAD特異的マーカーを生化学的に測定することによる物質の評価、
−タウオパシー、好ましくはADを治療および予防するための物質の検証系、
−タウオパシー、好ましくはADを検出するための診断マーカーおよびプローブの開発の検証系、
−高血圧、糖尿病、異脂肪血症および高コレステロール血症をタウオパシー、好ましくはADと組み合わせて治療するための物質の検証系を含む
アルツハイマー病を治療、予防、および診断するための物質のスクリーニングアッセイ系を提供する。
(図面の簡単な説明)
アルツハイマーtau発現ベクターの構築は以下を含む手順からなる:
− コーディング配列の設計、特に欠失およびトランケーションを1に示す、
− tau遺伝子のこれらの部分は適切な制限配列を備えたDNAポリメラーゼおよびオリゴヌクレオチドの校正の助けにより増幅されたPCRであり、該部分は真核性発現ベクター中に全般的または組織特異的プロモーターの下にクローンすることができ、クローニングは一般的細菌株を用い標準的手順を用いて行われる、
− 制限消化および/またはPCR分析により確認された挿入遺伝子の方向性を調べるためにクローン化遺伝子の確認を行った (図2A)。さらに、該構築物は考えられる突然変異型を排除するために部分的にシーケンシングした、
− マイクロインジェクションのための断片の精製 (図2B)、
− トランス遺伝子の哺乳動物細胞、主としてCOS-7およびC6ラットグリオーマ細胞へのトランスフェクションを用いるタンパク質発現の確認、およびwesternブロット法による真核性細胞におけるタンパク質発現の分析(図2C)。
実施例2:トランスジェニックラットの作製
例えばTg系統#318はトランス遺伝子4コピーを含み、これをSybre Green法およびBioRad PCR装置を用いるリアルタイムPCRにより決定した。
実施例3: フォスターマザーへの胚移植後に生まれた動物の遺伝子型別、トランスジェニック動物の同定およびトランス遺伝子の次世代への伝達の評価
遺伝子型別:二重トランケート型をコードするトランス遺伝子の特異的増幅をトランスジェニック動物の親世代由来のゲノムDNAについて行い、図3Aおよび3Bに示す。F1世代のゲノムDNAのさらなる分析は、トランス遺伝子が親世代の子孫にも同定されたことから遺伝性であることを証明した。したがってアルツハイマーtauをコードするトランス遺伝子は該動物の染色体DNAに固定される。遺伝子型別は図3Aに示すように2つの陽性(プラスミドおよびTG陽性ゲノムDNA)および2つの陰性コントロール(非増幅コントロールヘテロローガスゲノムDNA;化学物質のコントロール用の非テンプレートコントロール)を用いて行った。
トランス遺伝子発現の分析:トランス遺伝子由来のmRNAの発現をRT-PCRおよびアガロースゲル電気泳動を含む一般に知られた方法を用いるRT-PCR分析により評価した。RNAをトランスジェニック動物の瞬間凍結組織から抽出し、逆転写次いでcDNAの特異的増幅にかけた。
実施例4:トランスジェニックラットの脳におけるアルツハイマーtauタンパク質の発現
実施例5:トランスジェニックラット脳の免疫組織学的分析
組織を以下のごとく調製し、薄切した。トランスジェニックおよびコントロール動物をエーテルで麻酔し、速やかに緩衝4%パラホルムアルデヒドを心臓内に還流し、脳を取り出し、20%ショ糖で凍結保護し、急速凍結し、クリオスタットで50μmの厚さに薄切した。切片のいくつかをすぐに70%ギ酸で前処理し、次いで染色した。ラット脳組織切片をPBS中1%H2O2とインキュベーションし、非特異的結合部位をブロッキング培地(PBS、0,1% Triton、5%正常ウマ血清)中でインキュベーションしてブロックした。免疫染色を上記抗体を用いて実施し、次いでビオチン化ウマ抗マウス抗体、次いでアビジン-ビオチン複合体とインキュベーションし、次いでVIPおよびDAB溶液で可視化した。組織切片をゼラチンコートスライドにマウントし、乾燥し、段階的アルコール、キシレンで処理し、Entellanで封入した。
先に記載のGallyas銀染色法(23)を遊離浮遊50μm切片に適応し、金調色(gold toning)した。
間接免疫蛍光標識のため切片を1% NaHBO4で30分間前処理し、脳組織の自己蛍光を減少させた。次に、一次抗体で4℃で一夜染色し、ウマ抗マウスビオチン化抗体、次いでストレプトアビジン-Alexa488コンジュゲートとインキュベーションした。
実施例6:アルツハイマー病の治療および予防のための薬剤リードおよび薬剤候補のためのスクリーニングアッセイ系
(参考文献)
2. Wischik CM, Novak M, Edwards PC, Klug A, Tichelaar W, Crowther RA (1988a) Proc Natl Acad Sci USA 85: 4884-4888
3. Wischik CM, Novak M, Trogersen HC, Edwards PC, Runswick MJ, Jake R, Walker JE, Milstein C, Roth M, Klug A(1988b) Proc Natl Acad USA 85: 4506-4510
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Claims (13)
- 胚および/または体細胞がNおよびC末端トランケートtau分子の発現をもたらすコーディング領域を含むDNA構築物を含むトランスジェニック非ヒト動物であって、
-該コーディング領域が4反復配列および3反復配列tauタンパク質をコードする完全長tau cDNA配列の開始コドン下流の少なくとも30ヌクレオチドおよび終止コドン上流の少なくとも30ヌクレオチドがそれぞれトランケートされており、
-最小トランケートtauコアが配列番号9によりコードされるタンパク質断片を含み、
-該DNA構築物が該動物の脳細胞に発現すると神経原繊維的(NF)病状を生じる活性を有するタンパク質をコードするものである
該動物。 - 胚および体細胞が該DNA構築物を一時的または安定に発現し、脳のNF病状を示すことを特徴とする請求項1記載のトランスジェニック非ヒト動物。
- 該DNA分子によりコードされる該タンパク質が脳に発現する請求項1または2記載のトランスジェニック非ヒト動物。
- ラットである請求項1〜3のいずれかに記載のトランスジェニック非ヒト動物。
- 請求項1記載のトランスジェニックトランケートtauに特異的なオリゴヌクレオチドを用いる請求項1〜4のいずれかに記載のトランスジェニック動物の遺伝子型別法(遺伝子型判別法)。
- NF病状を発現し、ADに関連する危険因子、を生じさせる遺伝子背景を有することによりヒトの疾患モデルである請求項1〜4のいずれかに記載のトランスジェニック動物。
- ADの危険因子として高血圧を生じることができるADの動物モデルである請求項6記載のトランスジェニック非ヒト動物。
- ADの危険因子として糖尿病を生じることができるADの動物モデルである請求項6記載のトランスジェニック非ヒト動物。
- ADの危険因子として高コレステロール血症を生じることができるADの動物モデルである請求項6記載のトランスジェニック非ヒト動物。
- 物質の有効性または療法、具体的には神経原繊維的病状を減少させる療法を試験するためのin vivoアッセイとして請求項1〜4および6〜9のいずれかに記載の動物の使用。
- 該物質または療法が神経変性性疾患、具体的にはタウオパシー、好ましくはAD、および神経原繊維的病状に伴う他の神経変性性疾患のためのものである請求項10記載の使用。
- 請求項1記載の構築物を含む非ヒトトランスジェニック細胞系。
- 該細胞系がトランスジェニックラット胚由来のラット細胞系であることを特徴とする請求項12記載の細胞系。
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