JP2007502109A - 精原細胞を利用した鳥類キメラの生産方法及び鳥類キメラ - Google Patents
精原細胞を利用した鳥類キメラの生産方法及び鳥類キメラ Download PDFInfo
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Abstract
【選択図】図3
Description
この方法は、Ogawaら(1997)の方法及びその変形された方法により行われる。コラゲナーゼタイプIの溶解されたHBSS(Hank's Balanced Salt's solution)に前記精巣組織を添加して、一定時間反応した後、トリプシンで処理する。
コラゲナーゼタイプI、トリプシン、ヒアルロニダーゼII、及びDNase Iが溶解されたDMEM培地で前記精巣組織を分解する。
コラゲナーゼタイプI及びトリプシンの溶解されたHBSSで精巣組織を分解し、ピペッティングにより精巣組織の分解をさらに促進させる。
実験動物としては、韓国烏骨鶏と白色レグホーン種を使用し、各実験動物は、精巣から供与細胞の分離、及び分離された供与細胞の受容体精巣内への移植に利用された。
供与体である4週または24週齢の韓国烏骨鶏の精巣から細胞を分離し、これは、1994年Brinsterらの方法である2段階酵素方法(Two step enzymatic method)に基づき、鶏精巣の特性により若干の変形を加えた。
単一細胞に分離した後、移植前まで、体外で短期間培養した。期間は、0日、5日、10日及び15日であって、細胞は、幼い週齢(4週齢)と性成熟以後の週齢(24週齢)の精巣から分離された精巣細胞を利用し、1×108細胞を100mm細胞培養ディッシュに播いて、それぞれ5%CO2培養器で37℃に培養した。
分離あるいは体外培養された精原細胞は、遠心分離して2×107cells/50〜100μlに濃縮して、受容体精巣内に移植した。移植は、幼い週齢(7週齢)と成畜(24週齢)の受容体鶏に区分して進行し、全身麻酔のために、ケタミン注射液(YUHAN Corporation)を10mg/kg(20μl)翼静脈内に血管注射した。その後、麻酔された鶏の右側下の腹部を切開し、脊髄の下側に位置した精巣を確認した。精巣の確認後、用意した細胞浮遊液を針付注射器(Hamilton, 100 μl, 33G)を利用して精巣内に注入した。注入時、針の先は、精巣の外膜側に位置するが、これは、精巣細管の最も上側部分(upstream)に細胞を移植するためである。注入が終わった後、切開された腹部の内膜と外膜を、手術用縫合糸と針を利用して縫合し、手術部位を消毒した後、抗生剤を投与した。
鶏の解剖学的構造上、精巣は、腹腔内、脊椎の下に位置しているため、マウスにおける注入方式である手術を通じて精巣を露出させ、顕微鏡下で手術を行うことが難しい。したがって、この際は、注入針の太さと注入時の角度などが重要に作用する。したがって、分離された精巣に、同じゲージの注射針を利用して顕微鏡下でトリパンブルーを注入することにより、精巣細管内への細胞の注入が可能であるか否かを確認した。
受容体の白色レグホーンの精巣内に移植された韓国烏骨鶏の精原細胞から精子が形成されるかどうかを検証するために、検定交配を行った。白色レグホーン(I/I)は、黒色に対して優性であるため、黒色の烏骨鶏(i/i)と交配する場合、白色のヒヨコ(I/i)を生産するようになるが、受容体精巣内に移植された烏骨鶏の精原細胞由来精子と雌烏骨鶏の卵子とが結合する場合、正常形態である黒色の烏骨鶏ヒヨコ(i/i)を生産するようになり、これは、生殖腺キメラとして検証できる。
[精原細胞注入の確認]
鶏の解剖学的構造上、精巣は、腹腔内、脊椎の下に位置しているため、マウスにおける注入方式である手術を通じて精巣を露出させ、顕微鏡下で手術を行うことが難しい。したがって、この際は、注入針の太さと注入時の角度などが重要に作用する。したがって、分離された精巣に、実際の手術時使用されるものと同じゲージの注射針を利用して顕微鏡下でトリパンブルーを注入することにより、精巣細管内に細胞の注入が可能であるか否かを確認した。図2から、トリパンブルー溶液が精細管に沿って注入されることが確認できて、精巣全体に亘って注入がなされる様子を観察することができた。したがって、本研究で確立した手術方法と同一な注射針を利用した場合、受容体精巣内への精原細胞の注入が成功的になされることが分かる
生殖腺キメラの生産条件を確立するために、検定交配を行った。移植手術後、回復された2週後からの雌烏骨鶏と検定交配を行い、各実験区別に4匹ずつ組み込んだ。本実施例で利用された精原細胞は、5〜10日間体外培養したものである。
体外で短期培養した烏骨鶏精巣細胞の場合、15日間体外培養で細胞を維持することができた。図4及び5から分かるように、4週齢精巣細胞を体外培養した結果、安定的に維持することができ、15日以後には、群集(colony)を形成し、細胞数が増加することが分かった。24週齢の成鶏の精巣細胞を体外培養した結果も、4週齢と同様に、安定的に維持することができ、15日以後には、群集(colony)を形成し、細胞数が増加することが分かった。
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Claims (15)
- 次の段階を含む精原細胞を利用した鳥類キメラの生産方法:
(a) 供与体鳥類の精巣を収得する段階;
(b) 前記精巣から精巣細胞ポピュレーション(population)を分離する段階;
(c) 前記精巣細胞ポピュレーションを細胞成長因子の含まれた培地で培養して、精原細胞ポピュレーションを収得する段階;及び
(d) 前記培養した精原細胞ポピュレーションまたは前記精巣細胞ポピュレーションを受容体鳥類の精巣に注入してキメラを生産する段階。
- 前記段階(b)は、コラゲナーゼ、トリプシン、またはこれらの混合物を、前記収得した精巣の組織に処理して行われることを特徴とする、請求項1に記載の方法。
- 前記細胞成長因子は、繊維芽細胞成長因子、インシュリン様成長因子−1、幹細胞因子、及びこれらの組み合せからなる群から選択されることを特徴とする、請求項1に記載の方法。
- 前記培地は、分化抑制因子をさらに含むことを特徴とする、請求項1に記載の方法。
- 前記分化抑制因子は、白血病抑制因子であることを特徴とする、請求項4に記載の方法。
- 前記培地は、繊維芽細胞成長因子、インシュリン様成長因子−1、及び白血病抑制因子の混合物を含む補足物を含有することを特徴とする、請求項1に記載の方法。
- 前記培地は、血清及び抗酸化剤をさらに含むことを特徴とする、請求項1に記載の方法。
- 前記段階(d)は、精原細胞ポピュレーションまたは精巣細胞ポピュレーションを受容体の精巣細管に注入して行うことを特徴とする、請求項1に記載の方法。
- 前記段階(d)は、精原細胞ポピュレーションまたは精巣細胞ポピュレーションを受容体の精巣細管の最も上側部分に注入して行うことを特徴とする、請求項8に記載の方法。
- 前記鳥類は、鶏、鶉、七面鳥、鴨、鵞鳥、雉、または鳩であることを特徴とする、請求項1に記載の方法。
- 前記供与体及び受容体は、異種であることを特徴とする、請求項1に記載の方法。
- 前記段階(d)の後、検定交配を行って、精原細胞ポピュレーションの注入された受容体がキメラであるか否かを確認する段階をさらに含むことを特徴とする、請求項1に記載の方法。
- 供与体の精原細胞を精巣内に保有し、前記精原細胞から精子を形成する能力を有して、且つ、前記精子は、子孫に生殖腺転移される特性を有することを特徴とする、鳥類キメラ。
- 前記鳥類キメラは、請求項1〜11のいずれかに記載の方法により生産されることを特徴とする、請求項13に記載の鳥類キメラ。
- 次の段階を含む形質転換鳥類の生産方法:
(a)供与体鳥類の精巣を収得する段階;
(b)前記精巣から精巣細胞ポピュレーション(population)を分離する段階;
(c)前記精巣細胞ポピュレーションを細胞成長因子の含まれた培地で培養して、精原細胞ポピュレーションを収得する段階;
(c’)前記精原細胞ポピュレーションまたは前記精巣細胞ポピュレーションに外来遺伝子を転移させる段階;
(d)前記精原細胞ポピュレーションまたは前記精巣細胞ポピュレーションを受容体鳥類の精巣に注入する段階;及び
(e)前記受容体の子孫を得て、形質転換鳥類を生産する段階。
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