JP4095898B2 - 人工染色体を含むトランスジェニック動物のクローニング - Google Patents
人工染色体を含むトランスジェニック動物のクローニング Download PDFInfo
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Description
次に,大きな異種核酸構築物,例えば人工染色体を細胞中に導入して,安定なトランスフォームした細胞株および細胞を製造することができる。導入は,任意の適当な方法または方法の組み合わせにより行い,これには,限定されないが,マイクロインジェクション,細胞融合,ミクロセル融合,エレクトロポレーション,ソノポレーション,エレクトロフュージョン,発射衝撃,リン酸カルシウム沈殿,脂質媒介性移植システム,リガンド/レセプターシステムおよび当業者によく知られる他のそのような方法が含まれる。特に,ACEは,容易に単離して遺伝子デリバリーに用いることができる。これらの人工染色体はまた,遺伝子産物産生システム,遺伝的に形質転換されたヒト化動物臓器の製造,および,最も好ましくは,トランスジェニック有蹄動物の生成において用いることができる。
最も好ましくは,トランスジェニック動物は,異種核酸分子,好ましくは人工染色体を核ドナー細胞中に導入し,次にこの核ドナー細胞を除核したレシピエント細胞,最も好ましくは除核した卵母細胞中に融合させて核移植胚を形成し,この胚を活性化し,最後にこの胚を母体宿主に移植してトランスジェニック動物を生成することにより製造する。
さらに別の観点においては,本発明は,部分的には,本発明の全能性哺乳動物細胞から発出した任意の胚,胎児,および動物に関連し,ここで,これらの発生している細胞塊中の1またはそれ以上の細胞は,少なくとも1つの大きな異種核酸構築物,最も好ましくは人工染色体を含む。
本発明は,部分的には,少なくとも1つの人工染色体を含む全能性細胞に関する。これらの細胞は,クローン化トランスジェニック胚,胎児,および動物を樹立するために核移植法において使用することができる。これらの細胞および本発明の他の材料および方法は,クローン化トランスジェニック動物の樹立に向けた改良点である。
本発明は,部分的には,1またはそれ以上の人工染色体を含む全能性哺乳動物細胞を樹立することに関する。これらの細胞は,核移植法において核ドナーとして有用でありうる。これらの細胞を核移植法において利用することにより,クローン化されたトランスジェニックの胚,胎児,および動物を樹立する方法の効率を改良することができる。
多数コピーのいくつかのトランスジンを含む人工染色体発現系(ACes)を核移植技術を用いて製造したウシ胚中に取り込ませた。第1の方法においては,除核した卵母細胞中に核ドナー細胞とエレクトロフュージョンする直前にACesを直接注入することによりACesをウシ胚中に取り込ませて,サイブリッドを形成した。ACesの直接注入により,25%までの細胞がACesを含む胚盤胞が得られる。
人工染色体は,細胞内に導入することができるDNA分子を含む。DNA分子を細胞内に導入するための材料および方法は当該技術分野においてよく知られている。本発明は,1またはそれ以上の人工染色体を任意のタイプの細胞内に導入することに関する。細胞タイプの例は,本明細書において定義される。さらに,人工染色体は,細胞が,液体,組織,臓器,および動物中に存在する場合に,細胞内に導入することができる。
本発明は,部分的には,人工染色体を含む全能性細胞を利用するプロセスに関する。これらの細胞は,好ましくは,核移植プロセスにおいて核ドナーとして用いられ,ここで,核ドナーはレシピエント卵母細胞中に挿入される。核移植法は,典型的には核ドナーをレシピエント卵母細胞中に挿入する移動工程を含む。核移植法は,任意に融合工程(例えば,1またはそれ以上の電気パルスおよび/または1またはそれ以上の融合剤により行われる)を含み,任意に活性化工程(例えば,電気的刺激および/またはDMAPと組み合わせたイオノマイシン)を含むことができる。核移植法は,1またはそれ以上の核移植サイクルを含むことができ,各サイクル中の種々の工程は任意の順番で実施することができ,任意のサイクルにおいて2回以上反復することができる。
本発明は,人工染色体をウシ胚の核中に取り込ませる方法を記載する。これらの方法の1つにより,核移植ドナー細胞へのミクロセル媒介性染色体移植を使用して,90%より多くの細胞が細胞1個あたり1個のACesを含有するウシ胚盤胞が得られた。核移植ドナー細胞との融合の前に除核した卵母細胞にACesを注入する別の方法により,25%までの細胞が細胞1個あたり1個のACesを含有する胚盤胞が得られた。ACesを除核した卵母細胞中に注入することの潜在的利点は,ミクロセル融合および選択プロセスを回避することができるため,トランスジェニック胚をより迅速に生成することができることである。
サテライトDNAに基づく人工染色体の形成は先に記載されている(Kereso,,et al.1996)。ここに記載されるネズミACesは,約6000万塩基対(60Mb)のDNAを含み,セントロメア,テロメア,ネズミサテライト反復のブロック,および2つの領域の異種DNA,すなわちβ−ガラクトシダーゼをコードする5コピーのlacZ遺伝子およびハイグロマイシン耐性を付与する6コピーのハイグロマイシンホスホトランスフェラーゼ遺伝子を含むものであった("β−ACes")。
50−60日齢のウシ胎児からの生殖隆起細胞を以下のようにして単離した。胎児からの生殖隆起を3mg/mlのプロテアーゼ(Sigma#p6911)を含む2mlのTL−HEPES(Bio Whittaker #04−616F)中で滅菌した外科用ナイフを用いて細切した。細切した生殖隆起を37℃で40−50分間インキュベートし,次に2mlシリンジおよび25ゲージの針を用いて粉砕することにより解離させた。解離した生殖隆起細胞の懸濁液を15ml滅菌管中で10mlのTL−HEPESと混合し,300xgで10分間遠心分離した。上清を吸引した後,解離した細胞を10%FBS(Hyclone#A−111−D)および0.1mMβ−メルカプトエタノールを含む10mlのα−MEM培地(Gibco#32571−037)に再懸濁した。再懸濁した細胞を有糸分裂的に不活性化した胎児マウスフィーダー細胞を含む10本の培養フラスコ(75cm2)に等分に分け,各25ng/mlのウシ塩基性線維芽細胞成長因子(bFGF)およびヒト組換え白血病阻害因子(hrLIF)を補充したMEM培地(10ml)で培養した。
齧歯動物/ハイブリッド細胞株mM2CIからβ−ACesを有するミクロセルを製造し,先に記載されるように(Telenius,et al.,1999),若干の改変を加えて,ウシEG細胞に移植した。142x106個の分裂中期細胞を5つの50%パーコール(Pharmacia)クッション上にのせて,660x106個のミクロセルを得た。540x106個のミクロセルを1.25x108個のレシピエントEG細胞と融合させた。融合は37%PEG1450(Sigma)を用いて37℃で3分間行い,次に融合後18−24時間で0.125mg/mlハイグロマイシン−B(Calbiochem)中で薬剤選択を始めた。薬剤選択の期間は10−14日間であった。ハイグロマイシン耐性細胞は薬剤選択の開始の14−30日後に核移植に用いた。
ハイグロマイシン−耐性EG細胞の核移植は上述のようにして行った(およびStrelchenko,et al.2000に記載されるように)。除核した卵母細胞−ドナー細胞複合体をエレクトロポレーションにより融合させ,化学的に活性化し,7−10日間培養した。
β−ACesは,de Jongら(1999)にしたがってフローサイトメトリにより単離および精製したが,ただし,被覆緩衝液を改変した(10mM Tris−HCl pH7.5,0.1mM EDTA,100mM NaCl,30μMスペルミン,および70μMスペルミジン)(J−緩衝液)。β−ACesは,使用の直前に,回転バケットローターで2500xg,15分間,4℃の遠心分離により濃縮した。50μlを残してすべての上清を除去し,チューブを穏やかにたたくことによりペレットを再懸濁した。
透明帯(除核した卵母細胞−ドナー細胞複合体)の下に位置する,ドナー細胞を有する除核した卵母細胞を,10cmペトリ皿上の25μlの液滴のTL−HEPES培地中に入れた。10−20μlの濃縮β−ACesを別の液滴のJ−Buffer(25μl)中に入れた。次にこれらの液滴をミネラルオイルで覆った。
本明細書に記載されているようにして核ドナー細胞と除核した卵母細胞とを融合させた後,得られたサイブリッドを,10cmペトリ皿上でミネラルオイル下の一滴のTL−HEPES中に置いた。10−20μLの濃縮したβ−ACesを別の一滴のJ−緩衝液中に置いた。一個のβ−ACesをマイクロインジェクション針(Humagen#MIC10,7μmO.D.)中に吸い込み,針をサイブリッド形質膜の,好ましくはドナー細胞核の近くに押し出した。サイブリッド膜を,調節された負圧で卵母細胞膜が破裂するまで針に吸い込んだ。この工程によりピペットに吸い込んだ細胞質を,一個のβ−ACesがドナー細胞核に隣接しておかれるように,β−ACesを含む卵母細胞中に穏やかに放出した。
7−10日齢の拡張された胚盤胞の中期調製物は,これらを1μg/mlコルヒチンを含む培地中で12時間成長させることにより調製した。胚盤胞を,スライド上の30μlの2:1dH2O:培地中に5分間置いた。可能な限り多くの液体を除去し,透明帯が溶解し細胞が解離し始めるまで30μlの0.01MHCl/0.1%Tween−20を胚に加えた。1滴の冷固定剤(3:1メタノール:酢酸)を細胞上に滴加した。スライドを乾燥させ,少なくとも24時間ねかせ,その後にFISHを行った。胚盤胞を25cm2の培養フラスコ中で培地中で培養することにより拡大した胚培養物を生成し,これを胚盤胞と同様にして,ただし18ゲージの針および1mlのピペットを用いて,付着した培養物の一部を組織培養フラスコから取り出して,FISH用に調製した。
2回のミクロセル移植実験から,ハイグロマイシン−B耐性(hygR)EG細胞コロニー(>65)が得られた。HygR−EG細胞を核移植において核ドナー細胞として用い,136個のサイブリッドから33個の胚盤胞を得た(24%)。拡張した胚盤胞(21)を拡大した胚培養物中に置いて,細胞株を生成した。これらの胚細胞株の2つを,FISH分析を実施することができる点まで増殖させた。ある胚細胞株は90%より多い細胞で細胞1個あたり1個のβ−ACesを含んでおり,一方,他の細胞株は,90%より多い細胞で細胞1個あたり2個のβ−ACesを含んでいた。
β−ACesを140個の除核した卵母細胞−ドナー細胞複合体に注入し,得られた除核した卵母細胞−ドナー細胞複合体を,本明細書に記載されるようにして,融合させ,活性化し,培養した。β−ACesを注入したサイブリッドは,注入していないサイブリッドと同様の頻度(39%)で発生した。28個の胚盤胞をFISHで分析し,そのうち12個(43%)の胚盤胞がβ−ACesを含んでいた。トランスジェニック胚盤胞におけるモザイク現象は,1−25%の範囲であった(25%が1,10%が1,2%が4,1%が6)。
Claims (20)
- 除核したレシピエント卵母細胞中に核ドナー細胞を核移植することによりトランスジェニック有蹄動物胚を製造する方法であって、
(a)核ドナー細胞、またはその核を、核ドナー細胞と同じ種の除核したレシピエント卵母細胞と融合させて核移植胚を形成し、ここで、100キロ塩基対より大きな異種DNA分子が融合の前に前記卵母細胞または核ドナー細胞中に注入され、このことにより前記核移植胚は前記異種DNA分子を含み;そして
(b)核移植胚を活性化して前記トランスジェニック有蹄動物胚を得る、
の各工程を含む方法。 - 前記トランスジェニック有蹄動物胚を培養し、ここで前記培養工程が前記異種DNA分子の1またはそれ以上のマーカーについて選択することを含む、請求項1記載の方法。
- トランスジェニック有蹄動物が、ウシ、ヒツジ、ヤギ、およびブタからなる群より選択される、請求項1記載の方法。
- 異種DNA分子が、1またはそれ以上のテロメア、1またはそれ以上のセントロメア、および1またはそれ以上の複製起源を含む、請求項1記載の方法。
- 異種DNA分子が、トランスジェニック有蹄動物胚の細胞中の、同種DNAを本質的に含まない複製ユニット上に含まれる、請求項1記載の方法。
- 前記核移植胚が少なくとも二細胞期まで培養され、ここでトランスジェニック有蹄動物胚の細胞の少なくとも50%が異種DNA分子を含む、請求項1記載の方法。
- 核ドナー細胞が、体細胞、始原生殖細胞、胚性生殖細胞、および胚性幹細胞からなる群より選択される、請求項1記載の方法。
- 異種DNAが、少なくとも1つのトランスジンの複数のコピーを含む、請求項1記載の方法。
- 前記異種DNA分子が100キロ塩基対〜500メガ塩基対である、請求項1記載の方法。
- 前記異種DNA分子が人工染色体である、請求項1記載の方法。
- 請求項1記載のトランスジェニック有蹄動物胚からトランスジェニック有蹄動物を製造する方法であって、前記トランスジェニック有蹄動物胚を母体宿主中に移植して、完全胎児発生した胎児を生成し、そして,分娩させて前記トランスジェニック有蹄動物を生成することを含む方法。
- 前記移植工程の前に、前記トランスジェニック有蹄動物胚を少なくとも二細胞期まで培養する、請求項11記載の方法。
- トランスジェニック有蹄動物の細胞の少なくとも50%が異種DNA分子を含む、請求項11記載の方法。
- 請求項1記載のトランスジェニック有蹄動物胚からトランスジェニック有蹄動物を製造する方法であって、
(a)前記トランスジェニック有蹄動物胚を母体宿主中に移植して胎児を生成し;
(b)前記胎児から核ドナー細胞を取得し、ここで、前記細胞は前記異種DNA分子を含み;
(c)前記核ドナー細胞、またはその核を、前記細胞と同じ種の除核したレシピエント細胞と融合させて第2の核移植胚を形成し、ここで、前記第2の核移植胚は前記異種DNA分子を含み;
(d)前記第2の核移植胚を活性化して第2のトランスジェニック有蹄動物胚を生成し;そして
(e)前記第2のトランスジェニック有蹄動物胚を母体宿主中に移植して完全胎児発生した胎児を生成し、そして分娩させて前記トランスジェニック有蹄動物を生成する、
の各工程を含む方法。 - 前記移植工程の前に、前記トランスジェニック有蹄動物胚および/または前記第2のトランスジェニック有蹄動物胚を少なくとも二細胞期まで培養する、請求項14記載の方法。
- トランスジェニック有蹄動物の細胞の少なくとも50%が異種DNA分子を含む、請求項14記載の方法。
- 請求項1記載のトランスジェニック有蹄動物胚からトランスジェニック有蹄動物を製造する方法であって、
(a)前記トランスジェニック有蹄動物胚を母体宿主中に移植して胎児を生成し;
(b)前記胎児から1またはそれ以上の細胞を取得し、ここで、前記細胞の1またはそれ以上は前記異種DNA分子を含み、そして前記1またはそれ以上の細胞を培養して細胞培養物を取得し;
(c)前記細胞培養物から取得した核ドナー細胞、またはその核を、前記核ドナー細胞と同じ種の除核したレシピエント細胞と融合させて第2の核移植胚を形成し、ここで、前記第2の核移植胚は前記異種DNA分子を含み;
(d)前記第2の核移植胚を活性化して第2のトランスジェニック有蹄動物胚を生成し;そして
(e)前記第2のトランスジェニック有蹄動物胚を母体宿主中に移植して完全胎児発生した胎児を生成し、そして分娩させて前記トランスジェニック有蹄動物を生成する、
の各工程を含む方法。 - 前記移植工程の前に、前記トランスジェニック有蹄動物胚および/または前記第2のトランスジェニック有蹄動物胚を少なくとも二細胞期まで培養する、請求項17記載の方法。
- トランスジェニック有蹄動物の細胞の少なくとも50%が異種DNA分子を含む、請求項17記載の方法。
- 前記培養工程が前記異種DNA分子の1またはそれ以上のマーカーについて選択することを含み、このことにより前記細胞培養中の細胞の少なくとも90%が前記異種DNA分子を含む、請求項17記載の方法。
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WO2005040391A1 (en) * | 2003-10-27 | 2005-05-06 | Murdoch Childrens Research Institute | Compositions and methods for differentiating stem cells |
WO2009005844A1 (en) * | 2007-07-02 | 2009-01-08 | Gregory Aharonian | Methods for female mammalian spermatogenesis and male mammalian oogenesis using synthetic nanobiology |
US20090170203A1 (en) * | 2008-01-01 | 2009-07-02 | Aharonian Gregory P | Methods for female mammalian spermatogenesis and male mammalian oogenesis using synthetic nanobiology |
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US11377687B2 (en) | 2015-10-16 | 2022-07-05 | Inguran, Llc | Methods of genomic evaluation in livestock |
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