JP4512813B2 - 誘導的に骨細胞を欠失させることのできるトランスジェニック動物 - Google Patents
誘導的に骨細胞を欠失させることのできるトランスジェニック動物 Download PDFInfo
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Description
本発明は、トランスジェニック動物に関するものであり、より詳細には、誘導的に骨細胞を欠失させることのできるトランスジェニック動物に関するものである。
骨に関連する細胞としては、破骨細胞、骨芽細胞および骨細胞が知られている。破骨細胞や骨芽細胞の産生するタンパク質についてはいくつか知られており、そのうちの一部は骨代謝のマーカーとして臨床分野において利用されている。一方で、骨細胞(osteocyte)については、特異的な遺伝子産物は知られておらず、in vivoでの機能も解明されていない。骨細胞は、骨芽細胞が終末分化した細胞である。骨細胞は、休止期にあって増殖せず、硬組織に埋没している。そのため、細胞突起などの形態学的な特徴に基づいて骨細胞を単離することは可能と思われるが、その単離は極めて困難である。また、一般にin vitroでの遺伝子の発現パターンはin vivoとは異なるため、骨細胞の単離が可能であったとしても、in vivoにおける骨細胞の生物学的特性を分析することは困難とされている。
1.1 マウスDMP1遺伝子のプロモーター領域のクローニング
マウスDMP1遺伝子プロモーターを単離するため、マウスゲノムライブラリーから該遺伝子の転写開始点よりも5’側の配列を有するクローンをスクリーニングした。具体的には、Mouse genomic library Lambda FIX II(Stratagene社)からの1.2×106個のプラークを対象として、マウスDMP1遺伝子の第−3500〜−3000ヌクレオチド(500ヌクレオチド長:ヌクレオチドに付された番号は同遺伝子の翻訳開始コドン中のアデニン残基を1とした場合におけるそのヌクレオチド残基の位置を示す)をプローブとしてスクリーニングを行ない、その結果として14個の陽性クローンを得た。次いで、これら陽性クローンの全てのファージDNAを精製し、NotI消化により得られたDNA断片のヌクレオチド配列を決定した。その結果、最も長いプロモーター領域が含まれている2つのクローンを同定した。これらのクローンは、翻訳開始点(翻訳開始コドン中のアデニン残基)の300bp上流から転写開始点の9614bp上流までを含む同一のクローンであることがわかった(図1を参照)。
上述のスクリーニングにより得られたDMP1遺伝子プロモーター配列について、転写活性に必要な領域を、内在性のDMP1遺伝子を発現するマウス骨芽細胞株(MC3T3−E1細胞)を用いるルシフェラーゼアッセイ法によって調べた。
マウスDMP1遺伝子プロモーターの制御下においてジフテリア毒素のヒト型受容体を発現するDNA構築物を作製し、これにより付与されるジフテリア毒素感受性を確認した。
2.1 DNA構築物を含むマウスの作出
プラスミドベクターpDTR1.5kbおよびpDTR9.6kbを制限酵素SacII/ClaIで消化し、アガロースゲル抽出を行なうことにより、DNA構築物DTR1.5kbおよびDTR9.6kb(図3)を単離した。その後、これらDNA構築物のいずれかを、マウス受精卵へ注入した。
3系統のDTR1.5Tgマウスのうちの1系統(DTR1.5#1)および4系統のDTR9.6Tgマウスのうちの1系統(DTR9.6#1)について、HB−EGFの発現の有無を確認した。具体的には、DTR1.5#1マウスおよびDTR9.6#1マウスから各種主要臓器を取り出し、TRIzol Reagent(Invitorogen社:「TRIzol」はInvitorogen社の登録商標である)を用いて全RNAを抽出し、DNaseI処理により混入ゲノムDNAを消化した。次に、これらの全RNAを鋳型とするRT−PCRにより、HB−EGFのmRNA発現の有無を調べた。RT−PCRにおいて、DTR1.5#1マウスについては、HB-EGF-2F:5'-gtatccacggaccagctgctac-3'(配列番号12)およびHB-EGF-3R:5'-gggtccctcttcttccctagc-3'(配列番号13)からなるプライマーペアを用い、DTR9.6#1マウスについては、DMP1E1-F:5'-cgagaacttcgctgagg-3'(配列番号14)およびHB-EGF-2R:5'-gagggtccgggttgctggttcc-3'(配列番号11)からなるプライマーペアを用いた。
DTR1.5#1マウスおよびDTR9.6#1マウスに、ジフテリア毒素(Sigma社)を腹腔内投与するか、または関節から大腿骨骨髄腔に投与した。投与量は50μg/体重kgまたは500μg/体重kgとし、投与回数は1回または2回とした。最終投与の2日後に脛骨および大腿骨を取り出し、脱灰液(10%EDTA,pH7.4)に2週間浸して脱灰し、脱水した後にパラフィン包埋薄切標本を作製した。得られた骨標本をHE染色し、顕微鏡で観察することにより、骨細胞の死滅の有無を確認した。
Claims (9)
- 宿主に対して本質的に毒性を示さない化合物を投与することにより、該宿主において骨細胞を欠失させることを可能とするDNA構築物であって、
前記化合物に結合する受容体をコードするDNA、および該DNAに機能しうる形で連結されたDMP1遺伝子プロモーターを含んでなり、前記化合物と前記受容体との結合により該受容体を発現している細胞の死滅がもたらされるものであり、
前記DMP1遺伝子プロモーターが、DMP1遺伝子において転写開始点の5’側に隣接する少なくとも9.6kbpのプロモーター領域を含んでなるものであり、
前記受容体をコードするDNAと前記DMP1遺伝子プロモーターとの間に、DMP1遺伝子の第1イントロンを含んでなるものである、DNA構築物。 - 前記化合物がジフテリア毒素であり、前記受容体がジフテリア毒素受容体である、請求項1に記載のDNA構築物。
- 請求項1または2に記載のDNA構築物を含んでなる、ベクター。
- 請求項1または2に記載のDNA構築物または請求項3に記載のベクターを含んでなる、宿主細胞。
- 請求項1または2に記載のDNA構築物または請求項3に記載のベクターを含んでなる、トランスジェニック非ヒト動物。
- 前記DNA構築物がゲノム中に含まれている、請求項5に記載のトランスジェニック非ヒト動物。
- 前記非ヒト動物が生物実験に用いるための動物である、請求項5に記載のトランスジェニック非ヒト動物。
- 請求項5〜7のいずれか一項に記載のトランスジェニック非ヒト動物を製造する方法であって、目的とする非ヒト動物に請求項1または2に記載のDNA構築物または請求項3に記載のベクターを導入することを含んでなる、方法。
- 目的とする非ヒト動物への前記DNA構築物の導入が、該非ヒト動物からの受精卵に該DNA構築物を注入し、得られた受精卵を飼育することによって行なわれる、請求項8に記載の方法。
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