JP4551108B2 - Noc2ノックアウトマウス - Google Patents
Noc2ノックアウトマウス Download PDFInfo
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- JP4551108B2 JP4551108B2 JP2004110374A JP2004110374A JP4551108B2 JP 4551108 B2 JP4551108 B2 JP 4551108B2 JP 2004110374 A JP2004110374 A JP 2004110374A JP 2004110374 A JP2004110374 A JP 2004110374A JP 4551108 B2 JP4551108 B2 JP 4551108B2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/4702—Regulators; Modulating activity
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
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- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Description
Noc2-/-マウスの遺伝子ターゲティング:
本発明者等は、Noc2遺伝子のエキソンを3挟んだイントロン2の途中からイントロン3の途中までを置換することによって、Noc2欠損マウスを作り出した(図1)。すなわち、129Svマウスゲノムライブラリー(λDASHファージライブラリー中)をラットのNoc2コード領域の全長cDNA(配列番号1)(ヌクレオチド1〜1934)を用いてスクリーニングした。9個の陽性クローンを単離し、制限酵素による消化と、ラットNoc2全長cDNAをプローブとしたサザンブロット解析を組み合わせることにより、制限酵素マップを作成した。具体的には、ラットNoc2全長cDNAをプローブに、GenBankデータベースをサーチしたところ、マウスNoc2遺伝子をコードするゲノム配列が得られた(配列番号2)(ヌクレオチド1〜20492)。この配列データと、9個のゲノムクローンの制限酵素地図を比較することにより、エキソン1からエキソン3を含む、約15kbの制限酵素地図を作成した(図1)。
その結果、図2に示すように、Noc2-/-マウスでは、5.7kbに相当するバンドが消失し、代わりに5.1kbに相当するバンドが出現していた。野生型マウス(Noc2+/+)では5.7kbに相当するバンドのみが、またNoc2+/-マウスでは5.7kb及び5.1kbにそれぞれ相当するバンドが確認された。また、Noc2mRNAの発現欠如も下記のようにして確認した。図3に示すように、野生型マウスの下垂体及び副腎に検出されるNoc2 mRNAは、Noc2-/-マウスでは検出されなかった。更に、Noc2-/-マウスと野生型マウスの膵島からの総RNAを用い、Noc2遺伝子転写産物について下記のようにしてRT−PCRを行った。図4に示すように、Noc2遺伝子転写産物は、Noc2-/-マウスには全く検出されなかった。
標準的方法により、マウスの尾及び各種組織から、ゲノムDNA(サザンブロット用)及び総RNA(ノーザンブロット用)を調製した。
ゲノムDNAはSspI及びSphIで消化した。DNA(10μg)又はRNA(20μg)を1%アガロースゲルで電気泳動し、ナイロン膜に移しとった。高ストリンジェント条件で、32P標識プローブを用いてハイブリダイゼーションを行った。サザンブロットに用いたプローブは示したNoc2のゲノムDNA断片(NcoI〜SalI断片)(配列番号3)であった。ノーザンブロットに用いたプローブは、マウスNoc2のcDNA断片(配列番号4)(5’側非翻訳領域のヌクレオチド1〜41及び3’側非翻訳領域のヌクレオチド948〜994を含む)のヌクレオチド1〜994に対応する断片であった。
ノーザンブロットに用いたプローブは、上記マウスのNoc2のcDNA断片の両端に5'-CGAAGCAGATGTGACTCCTG-3'(配列番号5)、及び5'-TTCTGGAAGAGTTTGCCTCA-3'(配列番号6)の2種のプライマーを設定し、PCRで増幅し、アガロースゲルで電気泳動後、該当するバンドを切り出し、精製して用いた。なおマウスのNoc2 cDNA断片は、上記の2種のプライマーを用いて、マウス膵β細胞株であるMIN6細胞から抽出したRNAから作成したcDNAを鋳型にPCRで増幅し、pGEM-T easy ベクターにサブクローニングして作製した。
RT−PCRは、膵島の総RNA(10μg)を用いて行った。PCR産物の推定サイズは345bpである。PCRは30サイクル行った。PCRによる増幅に用いたプライマーはNoc2の順方向配列5'-GCAGTGGAAATGATCAGTGG-3'(配列番号7)及び逆方向配列5'-CATCACGTTCCTCTGCATTG-3'(配列番号8)のものである。
常法に従い、Noc2-/-親同士からの4分裂期の受精卵と、クラゲAequorea victoriaのグリーン蛍光タンパク質(GFP)を発現している野生型(Noc2+/+)の受精卵を合わせてキメラマウスを作成した。キメラ形成は、PCR又はゲノムサザンブロット法により判定した。GFPを発現している野生型の卵は、CAGプロモーターの調節下にGFPを発現している雄のトランスジェニックマウス(トランスジーンについてホモ接合)(26)を用いて準備した。すなわち、多数のGFP変異型の一つである増強型グリーン蛍光タンパク質(enhanced green fluorescent protein:EGFP)をコードするcDNAをPCRにより常法により増幅した。これらのプライマー中のEcoRI部位を、増幅したEGFPのcDNAをpCAGGS発現ベクター(ヒヨコのβ−アクチンプロモーター、サイトメガロウイルスのエンハンサー、β−アクチンのイントロン及びウシのグロビンポリアデニル化部位シグナルを含む)内に組み込むのに用いた。プロモーターからコード配列までを含む全インサートをBamHI及びSalIで切り出し、精製した。精製断片を野生型Noc2マウス受精卵に注射することにより、GFPトランスジェニックマウス株を作成し、仮親中に移植して発生させた。生後1日のマウスについて蛍光顕微鏡によりGFP発現の有無を調べ、これを発現しているマウスを選択した。
経口グルコース耐性試験は、16時間絶食させた12〜20週齢の雄性マウスで行った。浸水ストレス実験は、先に記載した(25)ところに準じて、グルコース負荷後に個々に拘束ホルダー中に固定し20℃の水(水深5cm)に垂直に15分間浸漬したマウスを用いて、実施した。血中グルコースレベルは、Antosense Glucose II (三共)を用いて全血で測定した。血清インスリンレベルは、超高感度ラットインスリンELISAキット(森永)によって測定した。
常法(27)により、コラゲナーゼ消化法で膵島を単離した。PTX(30ng/ml)の存在下又は不存在下に、RPMI1640倍地中で、膵島を48時間培養した。バッチインキュベーションは、先に記述したところに準じて行った(27)。培地中に放出されたインスリンは、ラジオイムノアッセイ(栄研化学)によって測定した。LacZ、Noc2wt(野生型)、又はNoc2AAA(変異型)のcDNAを有する組換えアデノウイルスを、メーカー(STRATAGENE)の説明書に従って作成した。Noc2AAAの作成ためには、先に記載したようにして(24)、Trp-Phe-Tyr(残基154〜156)を3個のアラニンで置換した。Noc2-/-マウスの膵島に、単離直後、これらのアデノウイルスを48時間感染させた。Flag標識したRab3のアイソフォームでトランスフェクトしたCOS−1細胞の細胞溶解液を、GTP−γSの存在下、GST−Noc2野生型(Noc2wt)又はGST−Noc2変異体(Noc2AAA)への結合につき評価した。
インスリン分泌実験も、新たに調製した膵島を培養することなく用いて行った。
コラゲナーゼ消化法により、膵臓腺房を調製した。アミラーゼ分泌実験は、Ohnishi et al. (28)に従って、僅かな改良を加えて行った。すなわち、単離した腺房を、先に記述したように(28)インキュベーション緩衝液〔10mM Hepes(pH7.4)、127mM NaCl、4.7mM KCl、0.6mM MgCl2、1.3mM CaCl2、0.6mM Na2HPO4、2.0mg/mlグルコース、Eagle’s MEMアミノ酸サプルメント、2mM L−グルタミン、1%BSA、0.01%大豆トリプシンインヒビターよりなる〕中に懸濁させ、37℃にて30分間プレインキュベートした。プレインキュベーションの後、腺房を遠心し、新たなインキュベーション緩衝液中に再懸濁させ、そして30pMのコレシストキニン(CCK)又は1μMのカルバコールの存在下若しくは不存在下に、37℃にてインキュベートした。インキュベーション中に上清に放出されたアミラーゼを、Amylase B-test WAKO (和光純薬工業)を用いて定量した。アミラーゼ分泌は、膵臓腺房細胞中の総細胞含量に対する培地への放出量として標準化した(アミラーゼ放出%として表示)。
何れも全長の、野生型Noc2及び変異型Noc2(Noc2AAA)をGST−融合タンパク質として発現させ、メーカー(AMERSHAM)の説明書に従って精製した。全長のRab3A、B、C及びD並びにRab5のcDNAを、pFLAG−CMV−2(SIGMA)中にサブクローニングした。共沈アッセイのためには、LipofectAMINE (INVITROGEN)を用いてCOS−1細胞を各プラスミドでトランスフェクトした。トランスフェクション後、細胞を、緩衝液中〔20mMのHEPES、pH7.4、200mMのNaCl、1mMのジチオスレイトール、5mMのMgCl2、1mMのATP及び0.26%(v/v)のCHAPS〕で超音波処理した。インビトロ結合アッセイは、先に記述した(15)ところに準じて行った。すなわち、FLAG標識したRab3アイソフォーム及びRab5でトランスフェクトしたCOS−1細胞からの細胞溶解液を、GDP−βS又はGTP−γSの存在下におけるグルタチオンビーズに固定化したGST−Noc2への結合について評価した。Rab3アイソフォームとRab5とGST−Noc2とは、抗Flag抗体による、又はラットNoc2に対するイムノグロブリンG精製抗体によるイムノブロットによって検出した。
膵臓と、消化管の種々の部分とを、野生型マウス及びNoc2-/-マウスから取り出し、0.1Mリン酸緩衝液、pH7.4中、4%パラホルムアルデヒド中で浸漬固定した。固定された組織を、慣用の手順で乾燥させてパラフィン包埋した。5μm厚のパラフィン切片を、分泌顆粒についてはヘマトキシリン及びエオシン(HE)、Azan又は過ヨウ素酸シッフ(PAS)で染色し、膵臓ホルモンについては免疫染色した。膵臓、胃及び唾液腺からの小さい組織切片を、2.5%のグルタルアルデヒド及び1%のOsO4で後固定し、エポキシ樹脂に包埋した。やや薄い及び超薄の切片を、トルイジンブルー及び酢酸ウラニル/クエン酸鉛で、光学顕微鏡及び電子顕微鏡による観察用に、それぞれ染色した。
Noc2遺伝子の破壊は、下垂体、副腎及び膵島からの総RNAのノーザンブロット分析又は逆転写酵素−ポリメラーゼ連鎖反応(RT−PCR)分析によって確認された。Noc2-/-マウスは正常に発生し繁殖力があり、全体的外観や挙動に何ら異常はみられない。Noc2は膵島のような内分泌組織おいて高レベルに発現されることから(16)、我々は先ず、内分泌膵臓機能を調べた。Noc2-/-マウスと野生型マウスとで、経口グルコース負荷後の血中グルコース及び血清中インスリンレベルは、正常な条件の下で少量の血液サンプルを採取したときには、同様であり、統計学的差はなかった(それぞれ、図5及び6)。しかしながら、より大量の血液を採取したときは、我々は偶然にも、野生型マウスはグルコース負荷後に共に正常なグルコースレベルとインスリン応答とを示すのに対し、Noc2-/-マウスは、野生型マウスに比して、低下したインスリン応答を伴った有意に高い血中グルコースレベルを示すことを見出した(データは示さず)。
膵臓ホルモンに対する免疫組織染色によってもまた電子顕微鏡分析によっても、Noc2-/-マウスの膵島の形態学にもインスリン分泌顆粒にも、明らかな異常は見られなかった。
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Claims (1)
- Noc2遺伝子の欠損に関してホモ接合体のマウスを準備し、これをストレス下におくことによる、グルコース負荷でインスリン応答の低下と血中グルコースレベルの上昇を示すマウスを得る方法。
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JP2004110374A JP4551108B2 (ja) | 2004-04-02 | 2004-04-02 | Noc2ノックアウトマウス |
US11/095,668 US7166764B2 (en) | 2004-04-02 | 2005-04-01 | Noc2 knockout mouse |
EP05007178A EP1586653B1 (en) | 2004-04-02 | 2005-04-01 | Noc2 knockout mouse |
DE602005008129T DE602005008129D1 (de) | 2004-04-02 | 2005-04-01 | Noc2 knockout Maus |
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JP2004110374A JP4551108B2 (ja) | 2004-04-02 | 2004-04-02 | Noc2ノックアウトマウス |
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US20090083865A1 (en) * | 2006-01-11 | 2009-03-26 | Chan Teh-Sheng | Transgenic Mouse Lines Expressing Human Ace2 and Uses Thereof |
EP2472262A3 (en) | 2007-07-10 | 2012-10-17 | Immune Disease Institute, Inc. | Stromal interacting molecule knockout mouse and uses thereof |
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JP2001136866A (ja) * | 1999-11-16 | 2001-05-22 | Kazuo Suzuki | 好中球走化性因子lect2遺伝子の機能が欠損したマウス |
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DE602005008129D1 (de) | 2008-08-28 |
US20060021073A1 (en) | 2006-01-26 |
US7166764B2 (en) | 2007-01-23 |
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