JP2004500038A - Nuclear transfer using selected donor cells - Google Patents

Abstract

The present invention provides a method for nuclear transfer by selecting and separating G1 cells from a donor cell population. The method defines the cell cycle stage of the donor nucleus and can produce transgenic or non-transgenic cloned embryonic cells, reconstituted embryos and whole animals for agricultural, pharmaceutical, functional food and biomedical applications. This is more advantageous than the prior art.

Description

【００７５】
実施例２．核移植後、シグマＢＳＡを補足した培地中で培養されたｉｎ　ｖｉｔｒｏ発生に及ぼすＧ０またはＧ１のいずれかに同調された濾胞ドナー細胞（Ｊ１およびＥＦＣ細胞系）の効果
７日培養後クローン化胚の胚盤胞期へのｉｎ　ｖｉｔｒｏ発生に関するＧ０およびＧ１濾胞ドナー細胞間の細胞周期段階の効果はないという知見を支持するさらなる証拠を表２に提供する。
【００７６】
２つの独立した濾胞細胞系により実験を実施した。
（１）「ＥＦＣ」；ニュージーランドフリージアン（Ｎｅｗ Ｚｅａｌａｎｄ Ｆｒｉｅｓｉａｎ）乳牛からの濾胞細胞系、および
（２）「Ｊ１」：ニュージーランドジャージー（Ｎｅｗ Ｚｅａｌａｎｄ Ｊｅｒｓｅｙ）未経産乳牛からの濾胞細胞系。
これらの実験において、ＥＦＣ細胞は細胞サイクルのＧ０においてのみ使用され、Ｊ１細胞はＧ１においてのみ使用された。しかし、双方の細胞系が濾胞細胞から由来し、再構成された胚が全て同一培地配合において培養されたことから、本実施例ではそのデータを組み合わせた。これらの実験において、これは、標準ＡｇＲ ＳＯＦ培地であったが、上記の実施例１のライフテクノロジーズＢＳＡではなくシグマ（Ｓｉｇｍａ）ＢＳＡ（シグマ化学会社（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏｍｐａｎｙ）；製品番号Ａ−７０３０）を補足した。
【００７７】
Ｊ１細胞は、有糸分裂終了後１〜３時間以内に融合されたドナー細胞で３〜６度の継代間で核移植に使用された。ＥＦＣ細胞を３〜８回の継代培養間で使用し、０．５％血清を含有する培地中９〜１８日間の培養によりＧ０期に同調させた。
【００７８】
表２に示されたｉｎ ｖｉｔｒｏの発生結果は、濾胞ドナー細胞では、Ｇ０細胞周期段階とＧ１細胞周期段階との間で胚盤胞へと発生する融合胚の比率に相違がないことを示す。
【００７９】
表２．細胞サイクルのＧ０またはＧ１のいずれかにおいて濾胞細胞（Ｊ１またはＥＦＣ）により再構成され、シグマＢＳＡを補足したＡＧＲ ＳＯＦ培地中で培養されたクローン化胚のｉｎ ｖｉｔｒｏ発生（平均±標準誤差）
【表２】

【００８０】
実施例３．核移植後のｉｎ　ｖｉｖｏ発生に及ぼすＧ０またはＧ１のいずれかに同調された濾胞ドナー細胞（Ｊ１およびＥＦＣ細胞系）の効果
図１に示すデータは、レシピエント牛への導入後の、細胞サイクルのＧ０またはＧ１のいずれかにおいて濾胞細胞により再構成されたクローン化ウシ胚の妊娠中の生存を示す。図１のデータは、上記の実施例１および２において具体的に説明した実験からまとめたものである。これには、２つの濾胞細胞系、すなわちＪ１とＥＦＣから生じたクローン化胚が含まれる。さらに、それには、シグマＢＳＡまたはライフテクノロジーズＢＳＡのいずれかを補足した外は、同じＡｇＲ　ＳＯＦ培地配合で生じた胚を含む。これら２つの濾胞細胞系のいずれの間にも後導入の生存能力に及ぼす効果がなく、また２つのＢＳＡ源の効果もないことから（胚盤胞への発生に対しては有意な効果があるが）、データをプールした。
【００８１】
図１のデータは、同調化したレシピエント牛の生殖器官に導入された、Ｇ０ドナー細胞により再構成された全部で８５個の胚、および早期Ｇ１ドナー細胞により再構成された全部で９５個の胚を表す。
【００８２】
図１は、通常の超音波検査法、直腸検査および自然分娩により測定された胚導入７日目から出産予定日までの妊娠を通して存在する胚／胎仔の百分率をプロットする。最も注目すべきことに、Ｇ１濾胞細胞を使用すると、月満ちて生存能のある子牛の誕生を生じた。Ｇ０ドナー細胞と比較すると、妊娠３０日から月満ちてから誕生するまでの全期間を通してＧ１細胞で再構成されたクローン化胚の胚生存は低くなる傾向があったが、しかし、このことはここに報告された導入数では統計的な有意性レベルに到達しなかった。
【００８３】
Ｇ１濾胞細胞では、導入された胚の９％（９／９５）は月満ちて子牛の誕生を生じた。しかしながら、これらの子牛のうち４頭は、出産時にまたは出産後まもなく死亡し、Ｇ１細胞からの生存能のあるクローン化牛の有効率は全体として５％（５／９５）となった。それに対して、Ｇ０ドナー濾胞細胞は、出産予定日まで２０％（１７／８５）発生を生じ、分娩中３頭の子牛が死亡して、生存能のある子牛は全体として１６％（１４／８５）の最高の有効率となった。
【００８４】
実施例４．核移植およびライフテクノロジーズＢＳＡを補足した培地中で培養後のｉｎ　ｖｉｔｒｏおよびｉｎ　ｖｉｖｏ発生に及ぼすＧ０またはＧ１に同調された、成牛雌の皮膚線維芽細胞（Ａｇｅ＋およびＡｇｅ−の細胞系）の効果
２つの独立した成牛皮膚線維芽細胞の１次細胞系での実験からのデータが組み合わされている。この２つの細胞系を「加齢＋」および「加齢−」と表示する。それらは共に、性熟期の遅い開始または早い開始のいずれかについてそれぞれ選択されたアンガス（Ａｎｇｕｓ）肉牛由来の雌細胞系である。ｉｎ ｖｉｔｒｏとｉｎ ｖｉｖｏ発生に関してこれら２つの類似細胞系の間に相違がないので、データは下記の表３および図２において組み合わせられている。
【００８５】
２つの細胞周期段階をこれらの実験で比較した。
（１）Ｇ１：ドナー細胞を有糸分裂終了後１〜３時間以内に細胞質体に融合した。
（２）Ｇ０：ドナー細胞を０．５％ＦＣＳを補足した培地中３〜１２日間培養した。レシピエント牛に導入されたこれら再構成胚について、細胞から４〜５日間血清を取り出した。
【００８６】
両方の細胞系からの細胞および両方の細胞サイクル処理を、７回の継代培養で核移植に用いた。再構成胚を、ライフテクノロジーズＢＳＡ（ライフテクノロジーズ製品番号３００３６−５７８）を補足した標準ＡｇＲ ＳＯＦ培地配合において培養した。
【００８７】
表３に示したデータは、Ｇ０細胞が低血清中にあった時間の長さの融合効率に関する効果を示すが、しかし、一旦細胞質体と融合すると、引き続くｉｎ ｖｉｔｒｏ発生に及ぼす効果がなかったので、Ｇ０胚盤胞データを組み合わせた。発生から胚盤胞期までに対するＧ０とＧ１との間の細胞周期段階の効果はなかった。
【００８８】
表３．細胞サイクルのＧ０またはＧ１で成牛メスの皮膚線維芽細胞（Ａｇｅ＋およびＡｇｅ−）により再構成され、ライフテクノロジーズＢＳＡを補足したＡｇＲ ＳＯＦ培地中で培養されたクローン化ウシ胚のｉｎ ｖｉｔｒｏ発生（平均±標準誤差）
【表３】

【００８９】
図２のデータは、Ｇ１において選ばれた成牛皮膚の線維芽細胞が月満ちて生存能のある子牛を産生できることを示している。図１のデータと同様に、Ｇ０では出産予定日での生存がより大きな傾向があるが、しかしこれは、ここで実施された導入数からは統計的に有意ではない。Ｇ１ドナー細胞では、導入された胚の４％（１／２５）が出産日に生存能のあるクローン化牛を産生した。それに対し、Ｇ０ドナー細胞は、出産日に１４％（３／２２）の生存能のある発生を生じた。本実験で、生まれた４頭の子牛全てが出生後生き残った。
【００９０】
実施例５．核移植およびＩＣＰ　ＢＳＡを補足した培地中培養後のｉｎ　ｖｉｔｒｏおよびｉｎ　ｖｉｖｏ発生に及ぼすＧ０またはＧ１に同調された、成牛皮膚線維芽細胞（ＬＪ８０１および３ＸＴＣ細胞系）の効果
２つの独立した成牛皮膚線維芽細胞系でＧ０およびＧ１細胞周期段階を比較する追加実験を実施した。この２つの細胞系は、「ＬＪ８０１」および「３ＸＴＣ」と表示した。ＬＪ８０１は、リモージンＸジャージー種のオス子牛由来のオス細胞系であり、３ＸＴＣは、３度において三つ子牛を出産した交雑育種牛由来である。
【００９１】
２つの細胞周期段階を比較した：
（１）両細胞系におけるドナー細胞を、０．５％ＦＣＳを有する培地中４〜５日間培養したＧ０細胞；および
（２）ドナー細胞を、有糸分裂後１〜３時間以内に細胞質体に融合したＧ１細胞。
【００９２】
負のＢｒｄＵ標識により、有糸分裂後３時間以内に融合した成牛皮膚の線維芽細胞がＳ期に入らなかったことが確認された。
ＬＪ８０１および３ＸＴＣの双方を、３〜４回の継代培養で核移植実験に用いた。
【００９３】
ＬＪ８０１細胞と３ＸＴＣ細胞との間で胚盤胞期への発生効率に差がなく、両細胞系から再構成された胚を、今回ＩＣＰ（イムノケミカルプロダクツ社、ニュージーランド国オークランド；製品番号ＡＢＦＦ−００２）からの別のＢＳＡ源を補足した同一の標準ＡｇＲ　ＳＯＦ培地中で培養したことから、表４のデータを組み合わせてある。胚導入後、妊娠を通しての胚生存に関するデータを、細胞系３ＸＴＣとＬＪ８０１とのそれぞれについて図３および４に示す。
【００９４】
表４のデータは、胚盤胞期への発生に対してＧ０またはＧ１のいずれかに同調されたドナー成牛皮膚線維芽細胞の細胞サイクルの効果が無かったことを示す。　表４．細胞サイクルのＧ０またはＧ１のいずれかでの成牛の皮膚線維芽細胞（ＬＪ８０１および３ＸＴＣ細胞）により再構成され、ＩＣＰ　ＢＳＡを補足したＡｇＲ　ＳＯＦ培地中培養されたクローン化胚のｉｎ　ｖｉｔｒｏ発生（平均±標準誤差）
【表４】

【００９５】
図３のデータは、３ＸＴＣ細胞により再構成された胚では、少なくとも１５０日目までの生存に差がなかったことを示す。細胞サイクルのＧ１におけるドナー細胞の１５０日目の胚生存は２５％（３／１２）であったのに対し、Ｇ０ドナー細胞では２７％（３／１１）であった。
【００９６】
図４のデータは、ＬＪ８０１線維芽細胞により再構成された胚では、Ｇ０ドナー細胞による妊娠２１０日目により大きな胚生存傾向があることを示すが、これは統計的に有意ではない。細胞サイクルのＧ１におけるドナー細胞では、２１０目における胚生存は、２３％（３／１３）であったのに対し、Ｇ０ドナー細胞では３９％（７／１８）であった。
【００９７】
実施例６．核移植後のｉｎ　ｖｉｔｒｏおよびｉｎ　ｖｉｖｏ発生に及ぼすＧ０またはＧ１のいずれかに同調された、遺伝的に変えた牛胎仔線維芽細胞の効果　本実験は、遺伝的に変えた牛胎仔メスの肺線維芽細胞で実施して、核移植後の発生に対する細胞周期段階の効果を調べた。遺伝子変更は、ウシβ遺伝子およびκ−カゼイン遺伝子の追加コピーの無作為挿入で行った。このトランスジェニック細胞系は、「カゼイン・プラス５１１０」と表示した。
【００９８】
２つの細胞周期段階を比較した：。
（１）ドナー細胞を、０．５％ＦＣＳを有する培地中３〜６日間培養したＧ０細胞；および
（２）ドナー細胞を、有糸分裂後１〜３時間以内に細胞質体に融合したＧ１細胞。
再構成胚をＩＣＰ　ＢＳＰ（イムノケミカルプロダクツ社、ニュージーランド国オークランド；製品番号ＡＢＦＦ−００２）を補足した標準ＡｇＲ　ＳＯＦ培地組成中で培養した。
【００９９】
ｉｎ　ｖｉｔｒｏ胚発生に関するデータを表５に示す。カゼイン・プラス５１１０細胞による胚盤胞期への発生は、核移植に使用したドナー細胞がＧ０における場合に比較してＧ１における場合は有意に少なかった。この結果は、前記実施例に示した他の細胞系と対照的である。
【０１００】
表５．細胞サイクルのＧ０またはＧ１期のいずれかでのトランスジェニックメスの肺皮膚線維芽細胞（カゼイン・プラス５１１０細胞）により再構成され、ＩＣＰ　ＢＳＡを補足したＡｇＲ　ＳＯＦ培地中培養されたクローン化牛胚のｉｎ　ｖｉｔｒｏ発生（平均±標準誤差）
【表５】

【０１０１】
図５に示した胚生存データは、カゼイン・プラス５１１０トランスジェニック細胞では、少なくとも９０日目までの発生に及ぼす細胞周期段階の効果が無いことを示す。細胞サイクルのＧ１におけるドナー細胞では、９０日目の胚生存は３８％（９／２４）であったのに対し、Ｇ０ドナー細胞では２７％（６／２２）であった。
【０１０２】
実施例７．核移植後のｉｎ　ｖｉｔｒｏおよびｉｎ　ｖｉｖｏ発生に及ぼす非増殖性老化ドナー細胞（後期Ｇ１期における）の効果
核移植実験を実施して、非増殖性老化牛ドナー細胞による発生に及ぼす効果を調べた。本実験に用いた細胞は、メス胎仔肺線維芽細胞であり、遺伝的に変更したものである（「５６１細胞」と表示される）。最初に、細胞は活発に増殖するが、しかし培養過程の間に、これは漸次遅くなり、後期継代時に細胞は、当業者により老化として知られている非増殖期に入り、それにより細胞分化が止まる。老化細胞は、細胞サイクルのＧ１、具体的には後期Ｇ１／Ｓ期境界時に止まっていることが知られている（Ｓｈｅｒｗｏｏｄ等、１９８８年）。細胞が老化に入る場合、それらは後期Ｇ１で遮断され、生理的有糸分裂促進因子に応答してＳ期に入ることがない。したがって、老化は静止期とは異なっている。静止期の細胞は至適条件（例えば、血清不足の細胞培養の場合、血清の追加）に戻すと、細胞サイクル再入へと誘導され増殖する。
【０１０３】
トランスジェニック老化細胞により再構成された胚を、ＩＣＰ　ＢＳＡを補足したＡｇＲ　ＳＯＦ培地中培養した。
ｉｎ　ｖｉｔｒｏ胚発生に関するデータを表６に示す。老化ドナー細胞による胚盤胞期への発生は、前記実施例に示したＧ０または早期Ｇ１ドナー細胞に関して予想された発生よりも低かった。
【０１０４】
表５．非増殖の老化トランスジェニック線維芽細胞（５６１細胞）により再構成され、ＩＣＰ　ＢＳＡを補足したＡｇＲ　ＳＯＦ培地中培養されたクローン化牛胚のｉｎ　ｖｉｔｒｏ発生
【表６】

【０１０５】
図６に示した胚生存データは、後期Ｇ１期に止まっている非増殖性老化細胞によるクローン化が、９０日目までに発生する胚がわずか４％（１／２６）である、という低効率であることを示唆する。
【０１０６】
（結論）
１．細胞周期のある特定の段階、好ましくは早期Ｇ１における個々の細胞を選ぶことが可能である。このことは、核移植に使用される個々の細胞における実際の細胞周期段階が正確に知られていない、いわゆる増殖細胞を用いる先行技術と比較して有利である。同じことが、血清飢餓にした細胞個体群にとっても言える。
【０１０７】
２．細胞サイクルの早期Ｇ１期における有糸分裂後の細胞は、生存牛の出産により立証されているように、核移植後全能性である。
３．このように、Ｇ０は、分化培養細胞を用いて核移植後の発生に適合する唯一の細胞周期段階ではなく、Ｇ１、好ましくは早期Ｇ１でも核は、機能的に再プログラム化できる。
【０１０８】
４．有糸分裂後の早期Ｇ１細胞は、核移植後の胚盤胞期への発生をＧ０における細胞と同様のレベルに促進する。
５．Ｇ０または早期Ｇ１ドナー核のいずれかにより再構成されたクローン化胚の出産予定日までの導入後生存において有意差はない。
【０１０９】
（産業上の利用可能性）
本発明は、特に治療的クローン化分野におけるヒトへの適用のみならず、製薬上有用な蛋白質、肉、牛乳、繊維などの農業的に有用な産物を生産できるトランスジェニック動物などの動物のクローン化群を確立するのに有用である。
【０１１０】
本明細書の記載が、上記実施例にのみ本発明の範囲を限定することを意図しているのではなく、添付の請求の範囲から離脱することなく、当業者により容易に生じる多くの変更が可能であることが解されよう。
【０１１１】
（引用文献）
Ｂａｒｎｅｓ Ｆ Ｌ； Ｃｏｌｌａｓ Ｐ； Ｐｏｗｅｌｌ Ｒ； Ｋｉｎｇ Ｗ Ａ； Ｗｅｓｔｈｕｓｉｎ Ｍ ａｎｄ Ｓｈｅｐｈｅｒｄ Ｄ （１９９３）。ＤＮＡ合成、核膜破壊、染色体構築および核移植ウシ胚における発生に及ぼすレシピエントの卵母細胞周期段階の影響。Ｍｏｌｅｃｕｌａｒ Ｒｅｐｒｏｄｕｃｔｉｏｎ ａｎｄ Ｄｅｖｅｌｏｐｍｅｎｔ ３６： ３３−４１。
Ｂｏｑｕｅｓｔ Ａ Ｃ； Ｄａｙ Ｂ Ｎ ａｎｄ Ｐｒａｔｈｅｒ Ｒ Ｓ （１９９９）。培養ブタ胎児線維芽細胞のフロー・サイトメトリによる細胞周期分析。Ｂｉｏｌｏｇｙ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ６０： １０１３−１０１９。
Ｃａｍｐｂｅｌｌ Ｋ Ｈ Ｓ； Ｌｏｉ Ｐ； Ｃａｐｐａｉ Ｐ ａｎｄ Ｗｉｌｍｕｔ Ｉ （１９９４）。除核活性化卵母細胞の予定Ｓ期の間に再構成されたヒツジ核移植胚の胞胚への改善された発生。Ｂｉｏｌｏｇｙ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ５０： １３８５−１３９３。
Ｃａｍｐｂｅｌｌ Ｋ； ＭｃＷｈｉｒ Ｊ； Ｒｉｔｃｈｉｅ Ｂ ａｎｄ Ｗｉｌｍｕｔ Ｉ （１９９５）。培養胚ディスク細胞の核移植後の生存子ヒツジの生産。Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ ４３： １８１。
Ｃａｍｐｂｅｌｌ Ｋ Ｈ Ｓ； Ｌｏｉ Ｐ； Ｏｔａｅｇｕｉ Ｐ Ｊ ａｎｄ Ｗｉｌｍｕｔ Ｉ （１９９６ａ）。核移植による胚クローン化における細胞周期調整。Ｒｅｖｉｅｗｓ ｉｎ Ｒｅｐｒｏｄｕｃｔｉｏｎ １： ４０−４６。
Ｃａｍｐｂｅｌｌ Ｋ Ｈ Ｓ； ＭｃＷｈｉｒｅ Ｊ； Ｒｉｔｃｈｉｅ Ｗ Ａ ａｎｄ Ｗｉｌｍｕｔ Ｉ （１９９６ｂ）。培養細胞系からの核移植によるクローン化ヒツジ。Ｎａｔｕｒｅ ３８０： ６４−６６。
Ｃｉｂｅｌｌｉ Ｊ Ｂ； Ｓｔｉｃｅ Ｓ Ｌ； Ｇｏｌｕｅｋｅ Ｐ Ｊ； Ｋａｎｅ Ｊ Ｊ； Ｊｅｒｒｙ Ｊ； Ｂｌａｃｋｗｅｌｌ Ｃ； Ｐｏｎｃｅ Ｄｅ Ｌｅｏｎ Ｆ Ａ ａｎｄ Ｒｏｂｌ Ｊ Ｍ （１９９８）。非静止期の胎児線維芽細胞から生産したクローン化トランスジェニック・ウシ。Ｓｃｉｅｎｃｅ ２８０： １２５６−１２５８。
Ｃｏｌｌａｓ Ｐ； Ｂａｌｉｓｅ Ｊ Ｊ ａｎｄ Ｒｏｂｌ Ｊ Ｍ （１９９２ａ）。核移植ウサギ胚の発生に及ぼすドナー核の細胞周期段階の影響。Ｂｉｏｌｏｇｙ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ４６： ４９２−５００。
Ｃｏｌｌａｓ Ｐ； Ｐｉｎｔｏ−Ｃｏｒｒｅｉａ Ｃ； Ｐｏｎｃｅ Ｄｅ Ｌｅｏｎ Ｆ Ａ ａｎｄ Ｒｏｂｌ Ｊ Ｍ （１９９２ｂ）。核移植ウサギ胚における染色質と紡錘体形態に及ぼすドナー細胞周期段階の影響。Ｂｉｏｌｏｇｙ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ４６： ５０１−５１１。
Ｃｚｏｌｏｗｓｋａ Ｒ； Ｍｏｄｌｉｎｓｋｉ Ｊ Ａ ａｎｄ Ｔａｒｋｏｗｓｋｉ Ａ Ｋ （１９８４）。不活化および活性化マウス卵母細胞における胸腺細胞核の挙動。Ｊｏｕｒｎａｌ ｏｆ Ｃｅｌｌ Ｓｃｉｅｎｃｅ ６９： １９３４。
Ｇａｄｂｏｉｓ Ｄ Ｍ， Ｃｒｉｓｓｍａｎ Ｈ Ａ， Ｔｏｂｅｙ Ｒ Ａ ａｎｄ Ｂｒａｄｂｕｒｙ Ｅ Ｍ （１９９２）。形質転換細胞において欠如する非形質転換哺乳類細胞のＧ１期における複数のキナーゼ停止点。Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅｓ， ＵＳＡ ８９： ８６２６−８６３０。
Ｇａｒｄｎｅｒ Ｄ Ｋ； Ｌａｎｅ Ｍ； Ｓｐｉｔｚｅｒ Ａ ａｎｄ Ｂａｔｔ Ｐ （１９９４）。血清と体細胞の不在下、胚盤胞段階までｉｎ ｖｉｔｒｏで培養したヒツジ接合子に関する卵割と発生の増大率：アミノ酸、ビタミンおよび胚の集団培養における発生刺激。Ｂｉｏｌｏｇｙ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ５０： ３９０−４００。
Ｐｅｄｅｒｓｅｎ Ｒ Ａ （１９９９）。医療のための胚幹細胞。Ｓｃｉｅｎｔｉｆｉｃ Ａｍｅｒｉｃａｎ Ａｐｒｉｌ １９９９： ４４−４９。
Ｐｅｒｒｙ ＡＣＦ， Ｗａｋａｙａｍａ Ｔ， Ｋｉｓｈｉｋａｗａ Ｈ， Ｋａｓａｉ Ｔ， Ｏｋａｂｅ Ｍ， Ｔｏｙｏｄａ Ｙ ａｎｄ
Ｙａｎａｇｉｍａｃｈｉ Ｒ （１９９９）。細胞質内精子注入による哺乳類の形質転換。Ｓｃｉｅｎｃｅ ２８４： １１８０−１１８３。
Ｓｃｈｎｉｅｋｅ Ａ Ｅ； Ｋｉｎｄ Ａ Ｊ； Ｒｉｔｃｈｉｅ Ｗ Ａ； Ｍｙｃｏｃｋ Ｋ； Ｓｃｏｔｔ Ａ Ｒ； Ｒｉｔｃｈｉｅ Ｍ； Ｗｉｌｍｕｔ Ｉ； Ｃｏｌｍａｎ Ａ ａｎｄ Ｃａｍｐｂｅｌｌ ＫＨＳ （１９９７）。形質移入胎児線維芽細胞の核移植により生産したヒトＩＸ因子トランスジェニック・ヒツジ。Ｓｃｉｅｎｃｅ ２７８： ２１３０−２１３３。
Ｓｈｅｒｗｏｏｄ Ｓ Ｗ， Ｒｕｓｈ Ｄ， Ｅｌｌｓｗｏｒｔｈ Ｊ Ｌ ａｎｄ Ｓｃｈｉｍｋｅ Ｒ Ｔ （１９８８）。ＩＭＲ−９０細胞における細胞老化の定義：フロー・サイトメトリによる分析。Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅｓ， ＵＳＡ ８５： ９０８６−９０９０。
Ｓｔｉｃｅ Ｓ Ｌ； Ｓｔｒｅｌｃｈｅｎｋｏ Ｎ Ｓ； Ｋｅｅｆｅｒ Ｃ Ｌ ａｎｄ Ｍａｔｔｈｅｗｓ Ｌ （１９９６）。多分化能ウシ胚細胞系による核移植後の胚発生の方向づけ。Ｂｉｏｌｏｇｙ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ５４： １００−１１０。
Ｓｕｓｋｏ−Ｐａｒｒｉｓｈ Ｊ Ｌ； Ｌｅｉｂｆｒｉｅｄ−Ｒｕｔｌｅｄｇｅ Ｍ Ｌ； Ｎｏｒｔｈｅｙ Ｄ Ｌ； Ｓｃｈｕｔｚｋｕｓ Ｖ ａｎｄ Ｆｉｒｓｔ Ｎ Ｌ （１９９４）。一過性カルシウム誘導後の蛋白質キナーゼ阻害により、減数分裂の完了なしでウシ卵母細胞は胚周期へ移行する。Ｄｅｖｅｌｏｐｍｅｎｔａｌ Ｂｉｏｌｏｇｙ １６６： ７２９−７３９。
Ｔａｄａ Ｍ， Ｔａｄａ Ｔ， Ｌｅｆｅｂｖｒｅ Ｌ， Ｂａｒｔｏｎ Ｓ Ｃ ａｎｄ Ｓｕｒａｎｉ Ｍ Ａ （１９９７）。胚生殖細胞による、雑種細胞内の体細胞核の後成的再プログラミング誘導。Ｔｈｅ ＥＭＢＯ Ｊｏｕｒｎａｌ １６ ： ６５１０−６５２０。
Ｔｈｏｍｐｓｏｎ Ｊ Ｇ Ｅ； Ｓｉｍｐｓｏｎ Ａ Ｃ； Ｐｕｇｈ ＰＡ； Ｄｏｎｎｅｌｌｙ Ｐ Ｅ ａｎｄ Ｔｅｒｖｉｔ Ｈ Ｒ （１９９０）。着床前のヒツジおよびウシ胚のｉｎ ｖｉｔｒｏ発生に及ぼす酸素濃度の影響。Ｊｏｕｒｎａｌ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ａｎｄ Ｆｅｒｔｉｌｉｔｙ ８９： ５７３−５７８。
Ｔｈｏｍｓｏｎ ＪＡ， Ｉｔｓｋｏｖｉｔｚ−Ｅｌｄｏｒ Ｊ， Ｓｈａｐｉｒｏ ＳＳ， Ｗａｋｎｉｔｚ ＭＡ， Ｓｗｉｅｒｇｉｅｌ ＪＪ， Ｍａｒｓｈａｌｌ ＶＳ ａｎｄ Ｊｏｎｅｓ ＪＭ （１９９８）。ヒト胚盤胞由来の胚幹細胞系。Ｓｃｉｅｎｃｅ ２８２ ： １１４５−１１４７。
Ｖｉｇｎｏｎ Ｘ； Ｃｈｅｓｎｅ Ｐ； Ｌｅ Ｂｏｕｒｈｉｓ Ｄ； Ｆｌｅｃｈｏｎ Ｊ Ｅ； Ｈｅｙｍａｎ Ｙ ａｎｄ Ｒｅｎａｒｄ Ｊ Ｐ （１９９８）。培養体細胞と融合した除核成熟卵母細胞から再構成されたウシ胚の発生能力。Ｃ Ｒ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅ， Ｐａｒｉｓ ３２１： ７３５−７４５。
Ｖｉｇｎｏｎ Ｘ； Ｌｅ Ｂｏｕｒｈｉｓ Ｄ； Ｃｈｅｓｎｅ Ｐ； Ｍａｒｃｈａｌ Ｊ； Ｈｅｙｍａｎ Ｙ ａｎｄ Ｒｅｎａｒｄ Ｊ Ｐ （１９９９）。静止および増殖皮膚線維芽細胞を用いて再構成したウシ核移植胚の発生。Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ ５１： ２１６。
Ｗａｋａｙａｍａ Ｔ ａｎｄ Ｙａｎａｇｉｍａｃｈｉ Ｒ （１９９９）． 成体尾先端細胞からの成体オス・マウスのクローン化。Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ２２： １２７−１２８。
Ｗａｌｌ ＲＪ， Ｋｅｒｒ ＤＥ ａｎｄ Ｂｏｎｄｉｏｌｉ ＫＲ （１９９７）。トランスジェニック乳牛：大スケールでの遺伝子工学。Ｊｏｕｒｎａｌ ｏｆ Ｄａｉｒｙ Ｓｃｉｅｎｃｅ ８０： ２２１３−２２２４。
Ｗｅｌｌｓ Ｄ Ｎ； Ｍｉｓｉｃａ Ｐ Ｍ； ＭｃＭｉｌｌａｎ Ｗ Ｈ ａｎｄ Ｔｅｒｖｉｔ Ｈ Ｒ （１９９８）。胎児線維芽細胞系の細胞による核移植後のクローン化ウシ胎児の生産。Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ ４９： ３３０。
Ｗｅｌｌｓ Ｄ Ｎ； Ｍｉｓｉｃａ Ｐ Ｍ； Ｔｅｒｖｉｔ Ｈ Ｒ ａｎｄ Ｖｉｖａｎｃｏ Ｗ Ｈ （１９９９）。最後のエンダービー・アイランド種の生き残りウシを保存するための成体体細胞核移植の使用。Ｒｅｐｒｏｄｕｃｔｉｏｎ， Ｆｅｒｔｉｌｉｔｙ ａｎｄ Ｄｅｖｅｌｏｐｍｅｎｔ １０： ３６９−３７８。
Ｗｉｌｍｕｔ Ｉ， Ｓｃｈｎｉｅｋｅ Ａ Ｅ； ＭｃＷｈｉｒ Ｊ； Ｋｉｎｄ Ａ Ｊ ａｎｄ Ｃａｍｐｂｅｌｌ Ｋ Ｈ Ｓ （１９９７）。胎児と成体哺乳類細胞由来の生存可能な子孫。Ｎａｔｕｒｅ ３８５： ８１０−８１３。
Ｚａｋｈａｒｔｃｈｅｎｋｏ Ｖ； Ｄｕｒｃｈｏｖａ−Ｈｉｌｌｓ Ｇ； Ｓｔｏｊｋｏｖｉｃ Ｍ； Ｓｃｈｅｒｎｔｈａｎｅｒ Ｗ； Ｐｒｅｌｌｅ Ｋ； ＳｔｅｉｎｂｏｒｎＲ； Ｍｕｌｌｅｒ Ｍ； Ｂｒｅｍ Ｇ ａｎｄ Ｗｏｌｆ Ｅ （１９９９）。ウシ胎児線維芽細胞を用いた核移植効率に及ぼす血清飢餓および再クローン化の影響。Ｊｏｕｒｎａｌ ｏｆ Ｒｅｐｒｏｄｕｃｔｉｏｎ ａｎｄ Ｆｅｒｔｉｌｉｔｙ １１５： ３２５−３３１。
全ての引用文献は参照により、その全体をここに組み入れている。
【図面の簡単な説明】
【図１】
ドナー細胞周期がＧ０またはＧ１のいずれかにある、濾胞細胞を用いて再構成したクローン・ウシ胚の妊娠期間中の生存率を示す図である。
【図２】
ドナー細胞周期がＧ０またはＧ１のいずれかにある、成体の雌の皮膚線維芽細胞を用いて再構成したクローン・ウシ胚の妊娠期間中の生存率を示す図である。
【図３】
ドナー細胞周期がＧ０またはＧ１のいずれかにある、成体の雌の皮膚線維芽細胞（３ＸＴＣ細胞）を用いて再構成したクローン・ウシ胚の妊娠期間中の生存率を示す図である。
【図４】
ドナー細胞周期がＧ０またはＧ１のいずれかにある、成体の雄の皮膚線維芽細胞（ＬＪ８０１細胞）を用いて再構成したクローン・ウシ胚の妊娠期間中の胚生存を示す図である。
【図５】
ドナー細胞周期がＧ０またはＧ１期にある、遺伝子的に改変された雌の胎児の肺線維芽細胞（カゼイン・プラス５１１０細胞）を用いて再構成したクローン遺伝子導入ウシ胚の妊娠期間中の胚生存を示す図である。
【図６】
非増殖老化雌胎児線維芽細胞（５６１細胞系統）を用いて再構成したクローン・トランスジェニック・ウシ胚の妊娠期間中の胚生存を示す図である。[0001]
The present invention relates to a novel method of nuclear transfer, particularly, but not exclusively, to genetically engineered or cloning techniques for the production of transgenic mammalian embryos, mammalian embryos, including fetuses and offspring, fetuses and offspring. .
[0002]
(background)
The cell cycle of the donor nucleus and the stage during embryo reconstitution of the recipient cytoplast are important factors for successful development after nuclear transfer. A specific combination of two "cells" is needed to ensure a diploid set of chromatin after the first goblet cell cycle and to maximize the chances of nuclear reprogramming and subsequent development Is done. When interphase donor cells fuse with metanuclear enucleated arrested oocytes (referred to as cytoplasts or recipient cells), immediate nuclear envelope destruction (NEBD) occurs and donor chromatin becomes immature chromosome condensation (PCC) (Barnes et al., 1993). These effects are triggered by cytoplasmic activity called oocyte maturation factor (MPF; also called meiosis or mitogen; see the review by Campbell et al., 1996a). MPF activity during oocyte maturation is maximal in the metaphase and falls off rapidly when subjected to either fertilization or artificial activation. Thus, two cytoplasts can be used for reconstitution. They are high or low in MPF using non-activated or activated metaphase II (MII) cytoplasts, respectively.
[0003]
Early work that promoted the understanding of the importance of cell cycle coordination in mammalian nuclear transfer to maintain chromosomal integrity and consequent developmental potential was based on transplantation in species such as rabbits, sheep, and cattle. This was performed using undifferentiated blastomeres (ie, undifferentiated embryo cells) obtained from a pre-stage embryo. These studies revealed that the cell cycle stage of the donor nucleus and the duration of exposure to MPF in the cytoplast had a significant effect on the degree of PCC observed. The chromatin of S-phase nuclei exposed to MPF usually presents a powdery appearance, which chromosomal studies have shown to be highly abnormal (Collas et al., 1992b). In contrast, when using G1 or G2 phase nuclei, chromatin condenses to form elongated chromosomes with single-stranded or double-stranded chromatids, respectively (Collas et al., 1992b). . After appropriate stimulation to release the reconstituted embryo from metaphase arrest and activate development, the nuclear membrane is reformed around the donor chromatin, which then reconstitutes with previous cell cycle stages. Perform DNA synthesis regardless. Thus, donor nuclei in the G1 phase initiate DNA synthesis compatible with normal development, while nuclei in the G2 or S phase, by completely or partially re-replicating the replicated DNA, to the first By the end of the goblet cell cycle, the DNA content in the two daughter cells is likely to be incorrect, resulting in abnormal early embryonic development.
[0004]
In contrast, from these earlier studies, NEBD does not occur (and thus PCC does not occur) at sufficient intervals from cytoplast activation and introduction of the blastomere nucleus into the cytoplast after the loss of MPF. It is the donor nucleus that controls DNA replication according to the cell cycle stage at the time of transfer. Thus, G1 or S phase nuclei, respectively, initiate or continue replication, while G2 phase nuclei are not induced to enter the next DNA synthesis. Such preactivated cytoplasts have been termed "universal recipients" (Campbell et al., 1994) and can regulate the development of donor cells at any stage of the cell cycle. This indicates that most undifferentiated blastomere nuclei are in S phase at any one time (80-90%; Barnes et al., 1993; Campbell et al., 1994) and are therefore most suitable for introduction into cytoploids with low MPF. It was especially important for cloning pretransplanted embryos.
[0005]
After nuclear transfer, normal development is in the oocyte cytoplasm, where factors (or extraneous additions) can reconstitute chromatin structures and reprogram gene expression patterns in the donor nucleus appropriately. Factor). The mature cytoplast contains RNA transcripts and proteins that govern the development of a normally fertilized cleavage-stage embryo until the time of normal genomic activation, and the embryo nucleus controls its own RNA synthesis that governs embryo development. Start on time. Thus, donor nuclei from embryos or cell types that have passed this point will cease RNA synthesis after reconstitution and remain inactive until a newly reprogrammed maternal-embryonic genomic transition occurs. Must-have. After transfer, the donor nucleus is forced to reprogram into a mating state, which then activates the appropriate gene at the correct level, timely and spatially appropriate for normal embryonic development to occur. The mechanisms for achieving such nuclear reprogramming are currently not fully understood.
[0006]
With the recent growing interest in performing nuclear transfer on cells that can be maintained in culture, reprogramming and cell cycle regulation have become important topics. These cultured cells are more differentiated (ie, have more individualized cell functions) than those used in earlier studies on cloning of embryonic blastomeres. These cells can be isolated from any embryo, fetus, or adult animal. As more cells are available, effective nuclear transfer techniques using these differentiated cell cultures enable large-scale propagation of the desired genotype in livestock species and genetic manipulation of the cultured cells It will facilitate the production of later transgenic animals.
[0007]
Early studies using asynchronously enriched cultures in which sheep embryo cells (cell cycle stage unknown) fused to preactivated cytoplasts proliferate early in passage from early passage cells (Campbell et al., 1995) Was actually born, but not from later passaged cells (Campbell et al., 1996b). Subsequent studies reported that cells were deprived of serum for 5 days and reported that the cells were in a quiescent phase (ie, the cells exited the normal cell division cycle and appeared to have entered the so-called "GO" phase). After fusion with the cytoplast before, after or at the same time as activation, live lambs were born with similar overall efficiency (Campbell et al., 1996b). These authors (Campbell et al., 1996b; Schnieke et al., 1997; Wilmut et al., 1997; WO 97/07669) propose that nuclear reprogramming be facilitated and that cloned animals be produced from differentiated cells. In addition, they suggest the importance of using cells that escape from the normal cell division cycle and are synchronized in the quiescent or G0 phase. A preferred method by the authors to synchronize cells to stationary phase (based on the absence of proliferating cell nuclear antigen (PCNA), indicating absence of cells in S phase) is by serum starvation for a suitable period of time. there were. Recently, however, questions have been raised as to what the percentage of serum-starved cells actually is in the G0 phase. Dual parameter flow method for simultaneously measuring cellular DNA and protein content (to distinguish G0 cells and G1 cells in a diploid population with quiescent cells having relatively little RNA and protein) Using cytometry, Boquest et al. (1999) examined cell cycle characteristics of cultured porcine fetal fibroblasts. They showed that, despite serum starvation for 5 days, less than 50% of cells were actually in G0 phase by their definition. By selecting "small" cells in the population, the percentage in the G0 phase was increased to 72% in serum-starved cultures (Boquest et al., 1999).
[0008]
As an alternative to the use of quiescent cells, Cibellelli et al. (1998; and WO 99/01163) fuse to the MII cytoplast and then use ionomycin and 6-dimethylaminopurine (6-DMAP). The use of non-serum starved random growth cultures of bovine cells activated by the method was reported. However, it is not clear from these disclosures what the cell cycle stage of the donor was at the time of nuclear transfer, in the embryos that ultimately produced cloned calves by these methods.
[0009]
Similarly, there are other reports in which cloned calves (Vignon et al., 1998, 1999; Zakhartchenko et al., 1999) and mice (Wakayama and Yamaguchi, 1999) were generated from nonserum-depleted random growth cell cultures. However, the cell cycle stage of the donor at the time of nuclear transfer that yielded cloned offspring is still unclear.
[0010]
Therefore, none of these previous studies accurately indicate which stage of the cell cycle has produced its low rate of reconstituted embryos that eventually develop into living offspring.
[0011]
The above considerations suggest that the identification of the cell cycle stage to which cultured donor cells belong during nuclear transfer has generally been inadequate so far, and the state of the art is uncertain and difficult to reproduce. Is clearly shown.
[0012]
Therefore, it would be desirable to have a nuclear transfer method that ensures that the cell cycle stage of the donor nucleus is known accurately.
It is an object of the present invention to approach the realization of this desire, or at least to provide the public with a useful choice.
[0013]
(Summary of the Invention)
According to a first aspect, the present invention provides a method for selecting and separating G1 cells from a growing or non-growing population of donor cells and introducing the separated nuclei of such G1 cells into enucleated recipient cells. And a method for nuclear transfer comprising: The donor cell population can be at one or more known or unknown stages in the cell cycle.
Such a method provides certainty regarding the cell cycle stage of the donor nucleus at the time of embryo reconstitution and is therefore advantageous over the prior art.
[0014]
The present invention contemplates the use of cell cycle inhibitors to block arbitrarily growing cells at specific stages of the cell cycle to produce a non-proliferative synchronized cell population. Preferably, cell growth is blocked at mitosis and cells that have progressed to the G1 phase after being released from the inhibitor are used for nuclear transfer.
[0015]
That is, in a second aspect, the present invention provides a method of nuclear transfer comprising introducing, into an enucleated recipient cell, a nucleus of a cell isolated from a non-proliferating cell population synchronized at the G1 stage of the cell cycle. .
[0016]
Preferably, said G1 cells are individually separated in the early G1 phase from an arbitrarily growing population or from a non-proliferating synchronized cell population.
Alternatively, the non-proliferating cell population can include senescent cells.
[0017]
The separated G1 donor cells are separated from the animal in vivo, more preferably from the cell culture in vitro. Suitable cells can be obtained from embryos, fetuses, young animals, to fully mature adults. In fact, any diploid karyotypic normal or senescent cell capable of cell growth can be used in the present invention. Cells can be in an undifferentiated cell state, or at any of a variety of cell differentiation stages, as long as they can be stimulated to enter the cell cycle and proliferate. Cells in the quiescent phase can be stimulated with appropriate culture conditions (such as the addition of serum or certain growth factors) into the cell cycle and used for nuclear transfer in the early G1 state following the mitotic phase. Some cell types may prove to be more effective than others, however, both adult and fetal fibroblasts, and adult follicular cells may be satisfactory. Have been found. As a demonstration of the present invention, results in bovine species using two follicular cell lines, four dermal fibroblast cell lines, and two genetically modified fetal fibroblast cell lines (Examples 1-7) Shown below.
[0018]
Preferably, the recipient cells include enucleated oocytes obtained from a species whose origin matches that of the donor nucleus. MPF activity during embryo reconstitution of enucleated oocytes may be high or low. This is because each state is compatible with the G1 donor nucleus having a 2C amount of DNA.
[0019]
Alternatively, the recipient cells can comprise enucleated stem cells, or clumps of enucleated stem cells fused together. Preferably, these stem cells are embryonic stem cells. Such embryonic stem cells used as recipient cells in nuclear transfer can be isolated from a developing embryo or from a stem cell line already established in culture. In this case, nuclear transfer using the donor nucleus of the G1 cell selected by the method of the present invention can be performed for the purpose of “therapeutic cloning”.
[0020]
According to a third aspect, the present invention provides a method for producing cloned animal embryos by introducing isolated donor nuclei in the G1, preferably early G1, phase into enucleated recipient cells.
[0021]
This method of the invention can be used to produce any animal embryo species of interest, including birds, amphibians, fish, and mammals. Preferably, the animal embryo of interest is a mammal, including but not limited to primates, including humans, rodents, rabbits, cats, dogs, horses, pigs, and most preferably bovine Includes ungulates such as, sheep, deer, and goats.
Preferably, the cloned animal embryo has the desired genetic trait by using a nucleus that has been genetically modified by methods known in the art.
[0022]
According to a fourth aspect, the present invention provides a reconstituted animal embryo prepared by the method of the present invention, comprising a reconstituted transgenic animal embryo. Embryos so formed can then be recloned to further increase the number of embryos or subjected to serial nuclear transfer to further promote nuclear reprogramming and / or developmental potential.
[0023]
According to a fifth aspect, the present invention provides (1) producing a cloned animal embryo according to the above-described method of the present invention, and (2) developing the animal from the embryo to a predetermined time by a known method, 3) Optionally, there is provided a method of cloning a non-human animal, including breeding an animal so formed by conventional or cloning according to the method of the present invention.
[0024]
The methods of the invention can be used to produce non-human animal species of interest, including birds, amphibians, fish, and mammals. Preferably, said non-human animal is from the group comprising non-human primates, rodents, rabbits, cats, dogs, horses, pigs, most preferably bovines, sheep, deer, and ungulates such as dogs. Selected.
[0025]
According to a sixth aspect, the present invention provides a non-human cloned animal prepared by the above-described method of the present invention, as well as the progeny of such a non-human cloned animal.
[0026]
The method of the present invention can be used to produce a non-human animal, preferably a mammal, having the desired genetic trait by using a donor nucleus that has been genetically modified by methods well known in the art. . Transgenic animals produced by such a method are also included in the present invention.
[0027]
The invention can further be used to produce embryonic cell lines, embryonic stem cells, fetuses, or progeny that can be used, for example, for cell, tissue, and organ transplantation.
[0028]
Thus, in a further embodiment, the present invention provides a method comprising: a) selecting and separating G1 cells from a growing population of donor cells in an unknown cell cycle, or a synchronized population of G1 cells, and enucleating the nuclei of such isolated cells. Introducing the cells into an ent cell, b) growing into the blastocyst stage, c) harvesting the embryonic cells, and d) establishing an infinite dividing cell line in vitro by methods known in the art. And methods for producing an embryonic cell line.
[0029]
In a further embodiment, a) selecting and separating G1 cells from a growing population of animal donor cells or synchronized cultures of G1 cells at various unknown stages of the cell cycle, and removing the nuclei of such separated cells from enucleated animals A method for producing animal embryonic stem cells is provided, comprising the steps of: introducing the cells into recipient cells; b) growing the cells at the blastocyst stage; and c) collecting the embryonic stem cells.
[0030]
Preferably, the animal donor cells used in the above method are of human origin. Most preferably, the animal donor cells and recipient cells used in the above method are both human.
[0031]
Most preferably, the donor cells are adult or fetal cells selected from any normal karyotype, and the recipient cells can reprogram gene expression, including enucleated oocytes or embryonic stem cells. It is selected from any possible cell type.
[0032]
Embryonic stem cells produced by the methods of the present invention are pluripotent, induced to differentiate in culture, and purified by methods known in the art, such as neurons, astrocytes, oligodendrocytes, etc. A pure population of differentiated forms of human cells can be formed, including hepatocytes, myocytes such as myocytes, heart cells such as cardiomyocytes, hematopoietic cells, pancreatic cells, and any other cell type.
[0033]
Such differentiated human cells and tissues can then be used for transplantation to treat certain diseases or injuries that the damaged cells cannot, or cannot replace effectively, by themselves. If human donor cells were obtained from a patient in need of such a transplant, such transplants would not be rejected by the patient because the tissue is genetically consistent with the patient.
[0034]
Alternatively, such differentiated cells and tissues may be diseased or injured, such as various neurological diseases (eg, Parkinson's disease), diabetes, heart disease, muscular dystrophy, various genetic diseases, certain cancers (eg, leukemia). , Spinal cord injury, burns, and other distresses.
These methods are known in the art as "therapeutic cloning".
[0035]
In vitro differentiation of human embryonic stem cells into specific cell types may also be beneficial in drug discovery and toxicity studies in human medicine.
[0036]
Thus, in a further aspect, the present invention provides a method of discovering a drug and testing the toxicity of the drug using the in vitro differentiated human embryonic stem cells produced by the method of the present invention.
[0037]
According to a further aspect, the present invention separates cells, tissues, and organs from non-human cloned animals and their progeny produced by the methods of the present invention and transplants them into a human patient in need of treatment. Xenograft methods that can be used for Where such cells, tissues, or organs contain a transgene, such cells, tissues, or organs may be useful in gene therapy or for suppressing a patient's immune response to xenogeneic tissue. .
[0038]
(Detailed description of the invention)
An object of the present invention is to improve a method for cloning a mammalian embryo by conventional nuclear transfer. Although the embryo cloning method of the present invention is intended to be used in various mammals and other animal species, the method will be described with respect to cattle. It is an essential feature of the present invention that the donor nucleus is in the G1 phase, preferably early in the G1 phase.
[0039]
One approach to reliably obtaining G1 cells from a population of loosely growing cultured cells is to individually remove cells in the mitotic phase from the culture surface and divide them in a medium containing 10% fetal calf serum (FCS). To complete it. Such donor cells are then fused with recipient cells (ie, cytoplasts) within a short time after division, for example, generally within 3 hours, before entering S phase (which can be detected by BrdU labeling). Let it. This ensures that the cells are in the early stages of G1 and have 2C of DNA. Thus, the cycle cells are used for nuclear transfer before proceeding to the G1 / S border. The selected cells are in a high serum-containing medium during this operation, at least until fusion with the cytoplast is completed. Thus, the cells are not induced to exit the cell division cycle and do not enter the stationary phase at any time.
[0040]
Although the present invention contemplates creating synchronized populations of G1 cells for use in nuclear transfer, an advantage of the method of the present invention is that many cells are pre-synchronized, eg, in M phase, and then the blocks are blocked. There is no need to use cytotoxic or cytotoxic cell synchronizing agents, such as nocodazole or colchicine, to release, allow cell division, and select cells in the early G1 phase. However, in the present invention, a drug suitable for selecting more G1 cells for use in nuclear transfer or for reversibly stopping cells at a specific point in the G1 phase, that is, a cell cycle inhibitor is used at an appropriate drug concentration. And it may be advantageous to use it for the incubation time. Such agents and methods will be known to those skilled in the art. These methods may be pre-tuned to reduce drug exposure time: induce cells to enter G0, eg, by temporarily removing serum, and then re-adding serum, The cells are returned to the G0 / G1 boundary or R point (restriction point) and the cell division cycle is advanced. Suitable agents for reversibly arresting cells at various points in G1 (Gadbois et al., 1992, and references therein) include (1) staurosporine (a very small molecule in the nanomolar region). (3) non-specific kinase inhibitors used at low concentrations), (2) kinase inhibitors more specific for cell cycle progression, such as cAMP-dependent protein kinase, and cGMP-dependent protein kinase inhibitors. Lovastatin, (4) isoleucine deficiency, (5) aphidicolin or hydroxyurea used to prevent entry into S phase and block cells at the G1 / S border.
[0041]
The G1 donor nucleus has a 2C amount of DNA (ie, is diploid) and can therefore be reconstituted with cytoplasts with high or low MPF levels. That is, the G1 nucleus can be introduced into the cytoplast before, after, or simultaneously with activation. However, in order to improve the development of cloned embryos, donor nuclei (either introduced by electrically induced cell fusion or introduced by direct nuclear injection) should be used to promote nuclear reprogramming. Exposure to an element present in the cytoplasm of the enucleated oocyte is preferred for a suitable time. This is called "pre-activation fusion" or FBA. Previous studies have demonstrated essentially "fusion at the same time as activation", ie, the advantage of this approach when compared to AFS (Stice et al., 1996, Wells et al., 1998, 1999). . Furthermore, it is recommended that exposure to the cytoplasm be at least greater than 1 hour, preferably between 3 and 6 hours, in order to increase the incidence of blastocysts. However, in this method, it is important that micronuclei formation, which occurs when fusion precedes activation, is prevented by any suitable means, in order for the resulting embryo to maintain correct ploidy (Czolowska et al., 1984).
[0042]
The following outlines the method of reconstitution and production of cloned embryos in bovine species obtained from G0 (control) and G1 cultured donor cells. In the specific examples described below, bovine follicle cells collected from follicles were used, and the results are shown in Examples 1 to 3 (the use of a fibroblast cell line is exemplified in Examples 4 to 7). In fact, essentially any cell type that has a normal diploid karyotype (embryonic, fetal, immature, and adult cells, is capable of proliferating or entering the cell cycle, or aging Cells in phase) are also demonstrated to be totipotent by using this technique. Further, as will be appreciated by those skilled in the art, any other method known in the art can be used to reconstitute and produce cloned embryos.
[0043]
Oocyte maturation in vitro
Ovaries were harvested from slaughterhouse cows, placed in saline (30 ° C.) and transported to the laboratory within 2 hours. Cumulus-oocyte complex (COC) was recovered by aspirating 3-10 mm follicles using an 18 gauge needle and negative pressure (alternatively, immature oocytes collected from donor bovines). May be collected and then matured in vitro). The COC is a Hepes buffered tissue culture medium (HEPES) supplemented with 50 μg / ml heparin (Sigma, St. Louis, MO) and 0.4% w / v BSA (Immuno-Chemical Products (ICP), Auckland, New Zealand). Tissue Culture Medium) 199 (H199, Life Technologies, Auckland, New Zealand). Prior to in vitro maturation, only COCs with compact non-retracted cumulus-radiation crowns and homogenous egg cytoplasm were selected. It was washed twice with H199 medium + 10% FCS (Life Technologies) and then once with bicarbonate ion buffered tissue culture medium 199 medium + 10% FCS. Ten COCs were transferred into 10 μl of this medium, which was placed in droplets of 40 μl of mature medium in a 5 cm Petri dish (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) and paraffin oil (Squibb, Princeton, Princeton). NJ). The maturation medium contained 10% FCS, 10 μg / ml sheep FSH (Ovagen, ICP), 1 μg / ml sheep LH (ICP), 1 μg / ml estradiol (Sigma), and 0.1 mM cystamine (Sigma). Supplemented Tissue Culture Medium 199. The culture dish containing the droplets is humidified in a 5% CO ₂ And cultured at 39 ° C. After maturation, COC was vortexed for 3 minutes with 0.1% hyaluronidase (bovine testis, Sigma) in HEPES buffered Synthetic Oviduct Fluid (HSOF, Thompson et al., 1990) followed by HSOF + 10%. The cumulus-radiation crown was completely removed by washing three times in FCS.
[0044]
Nuclear transfer using cultured cells
a) Medium Mature oocytes, cytoplasts, and reconstituted embryos were maintained or manipulated in H199-based medium after maturation until 15-30 minutes after electrical pulse to evaluate fusion. Embryos reconstituted by fusing MII cytoplasts with donor cells 3-6 hours prior to activation (FBA treatment) were used to transform calcium-free AgResearch Synthetic Ovidduct Fluid medium (AgRSOF, which is Gardner et al., 1994). A modified formulation of the described medium, commercially available from AgResearch, Hamilton, New Zealand) + 10% FCS for approximately 3-6 hours until just prior to activation. Hereinafter, all the medium compositions used contain calcium.
[0045]
b) Enucleation Oocytes matured for about 18-20 hours were enucleated by aspirating the first polar body and the MII plate with a small amount of surrounding cytoplasm with a 15-20 μm (outer diameter) glass pipette. The oocytes were previously stained with H199 medium containing 10% FCS, 5 μg / ml Hoechst 33342, and 7.5 μg / ml cytochalasin B (Sigma) for 5 to 15 minutes, and the operation did not include Hoechst 33342. Performed in the same medium. Enucleation was confirmed by visualizing the nucleoplasm with ultraviolet light. Following enucleation, the resulting cytoplasts were washed well with H199 + 10% FCS and kept in this medium until the injection of donor cells.
[0046]
c) Preparation of Stationary Phase (G0) Donor Cells Cultured follicle cells were induced to enter the stationary phase by eliminating serum (Campbell et al., 1996b). One day after normal subculture, the medium was aspirated off and the cells were washed three times with PBS each time, followed by addition of fresh medium containing only 0.5% FCS. Follicle cells were cultured for an additional 9-23 days (usually 10 days) before use for nuclear transfer. Immediately before injection, a single cell suspension of donor cells was prepared by standard trypsinization. Cells were pelleted, resuspended in H199 + 0.5% FCS, and kept in this medium until injection.
[0047]
d) Preparation of G1 donor cells Growing follicle cells were cultured on cover slips in culture dishes in a suitable medium (eg, DMEM / F12 + 10% fetal calf serum). The coverslip, on which the cells are growing on the surface, is aseptically removed physically from the culture dish and placed in a suitably configured micromanipulation chamber so that the cells or nuclei can be collected and injected. The cells are covered with a drop of HEPES buffered medium containing 10% FCS and then with mineral oil. Dividing cells are rounded and only loosely adhere to the medium surface, so unless the density of cells on the coverslip is too high, identify dividing cells and carefully select physically from the coverslip with an operational pipette. Can be. If this is difficult, wash the cells briefly with PBS and remove 1.5 μg / ml cytochalasin B, mainly to minimize mechanical damage caused by physically removing the cells from the coverslip. (For example, 1/10 of the concentration used for normal subculturing of a cultured cell line). Using a suitable microscope (eg, a phase contrast microscope, or a DIC light microscope), mainly by looking at concentrated chromatin on the mitotic spindle, or cells still connected by a cytoplasmic bridge at the late stages of division By confirming the concentrated chromatin in the doublet, dividing cells on the coverslip can be identified. In this way, it is not necessary to expose the cells to UV light using a DNA-specific fluorescent dye such as Hoechst 33342. Utilizing an injection pipette mounted on the manipulator, these dividing cells are separately removed from the coverslip and the adjacent 10% cells are allowed to complete and eventually divide to form a doublet of cells. Place in droplets of HEPES buffer medium containing FCS. The diameter of the pipette must be appropriate for the cell lineage so that the cells or spindle are not physically damaged during the operation. Thus, dividing cells are individually selected, preferably at late or late stages, and removed to complete mitosis and divide into two. The cell doublet is then gently separated into individual cells, which can be easily achieved by brief exposure to the appropriate enzyme solution. Thereafter, each intact cell is injected into the cytoplast and fused. Alternatively, the cell nucleus can be isolated and injected directly into the cytoplasm of enucleated oocytes. Preferably, the introduction of the donor nucleus into the cytoplast is completed within 3 hours of first removing the dividing cells from the culture surface. This ensures that the cultured cells are fused during the early GI phase of the cell cycle, but before the S phase. This should be confirmed for the particular cell type or cell line used, for example, by a negative BrdU labeling of a sample of the selected cells. It is further recommended to confirm that the cells selected by this method are indeed in the cell cycle and enter the S phase at some later point in time.
[0048]
e) Microinjection Recipient cytoplasts were dehydrated in H199 containing 10% FCS, and 5% sucrose. This medium was also used as a medium for micromanipulation. An appropriately sized pipette (eg, 30-35 μm OD) containing the donor cells is inserted through the zona pellucida and the cells are brought into contact with the zona pellucida and the cytoplast to bring the membrane into intimate contact for subsequent fusion. Pressed between membranes. After injection, the reconstituted embryos were rehydrated in two stages: First rehydrated in H199 containing 10% FCS and 2.5% sucrose for 5 minutes, then in H199 + 10% FCS until fusion.
[0049]
f) Cell fusion For both GO and Gl cell treatment, embryos were reconstituted using the FBA (pre-activation fusion) method. The reconstituted embryos were placed in a buffer containing 0.3 M mannitol, 0.5 mM HEPES, and fatty acid free (FAF) containing 0.05% BSA, 0.05 mM calcium, and 0.1 mM magnesium approximately 24 hours after the onset of maturation. At time (hpm), the electrofusion was performed. Fusion was performed at room temperature in a chamber with two stainless steel electrodes 500 μm apart covered with fusion buffer. The reconstituted embryos were manually aligned using a thin, mouth-operated Pasteur pipette so that the contact surface between the cytoplast and the donor cells was parallel to the electrodes. In the case of follicular cells, cell fusion was induced by two DC pulses of 15 μs and 2.25 to 2.50 kV / cm each from BTX Electrocell Manipulator 200 (BTX, San Diego, CA) (each cell). Optimal electrical parameters need to be determined for the system). After electrical stimulation, reconstituted embryos were washed in H199 + 10% FCS. Then, within 15-30 minutes, the fusion was confirmed under a microscope.
[0050]
The electrofusion parameters described above are not expected to result in significant rates of activation in young cytoplasts used at 24 hpm. Less than 1% of control oocytes of the same age (n = 112) formed pronuclei after similar electrical stimulation (Wells et al., 1999). It is important for FBA treatment that NEBD and PCC occur, exposing the G1 and G0 nuclear donor chromatin to elements present in the cytoplasm of the oocyte to allow chromatin remodeling, nuclear reprogramming It is.
[0051]
Alternatively, the nuclei of the cells of G1 can be isolated and injected directly into the cytoplasm of the oocyte, as known to those skilled in the art.
[0052]
g) Activation Artificial activation can be provided in a variety of ways. One particular method involves the combination of ionomycin (Sigma) and 6-dimethylaminopurine (6-DMAP, Sigma) (Susko-Parrish et al., 1994). After fusion, the embryo is activated, preferably after the donor nucleus has been exposed to the oocyte cytoplasm for 3-6 hours. This preferred method is called "pre-activation fusion" (FBA, Wells et al., 1998). Thirty minutes before activation, FBA-treated fused embryos were washed and maintained with HSOF (containing calcium) + 1 mg / ml FAF BSA. Activation was induced by incubating the embryos in 30 μl drops of 5 μM ionomycin (Sigma) in HSOF + 1 mg / ml FAF BSA for 4 minutes at 37 ° C. Activation generally occurs in the cytoplast at 27-30 hpm. The embryos are then thoroughly washed in HSOF + 30 mg / ml FAF BSA for 5 minutes, followed by 4 mM in AgRSOF (with calcium) + 10% FCS for 2 hours in 2 mM 6-dimethylaminopurine (6-DMAP, Sigma). And cultured.
[0053]
As in the FBA methodology (Stice et al., 1996, Wells et al., 1998, 1999), such delays increase the time of exposure of the nucleus to the oocyte cytoplasm and improve embryonic development rates. Must be combined with appropriate treatments to prevent the formation of micronuclei as a result of activated activation (Czorowska et al., 1984). Serine-threonine kinase inhibitors such as 6-DMAP are considered to be one suitable drug. Thus, 6-DMAP allows for the formation of a single intact nucleus after the initial activation stimulus, thus maintaining the correct ploidy in reconstructed embryos.
[0054]
In vitro culture of nuclear transfer embryos
Embryo culture was performed in 20 μl droplets of AgR SOF (commercially available from AgResearch, Hamilton, New Zealand) covered with paraffin oil. AgR SOF is a modified formulation of SOFaa BSA (containing 8 mg / ml FAF BSA, described in Gardner et al., 1994). When possible, groups of up to 10 embryos were cultured together in droplets of medium. Embryos were incubated in a humidified modular incubator chamber (ICN Biomedicals, Aurora, OH) at 39 ° C., 5% CO 2. ₂ : 7% O ₂ : 88% N ₂ Culture was performed in a mixed gas. On the fourth to fifth day of development (Day 0 = embryo reconstitution day), as taught in published patent specification WO 00/38583, which is hereby incorporated by reference, the incorporation of bovine embryos To improve in vitro generation, it was transferred to a fresh 20 μl droplet of 10 μM 2,4-dinitrophenol + AgRSOF, which acts as an uncoupler for the oxidative phosphorylation reaction. On the seventh day after the fusion, it was evaluated whether the cells had developed into blastocysts of transplantable quality.
[0055]
Embryo transfer, conception diagnosis, and childbirth
Embryo transfer, conception diagnosis, and delivery management were performed using techniques well known in the art.
[0056]
Serial nuclear transfer and recloning
In a further embodiment of the invention, it may be desirable to reclone the first generation cloned embryos created by reconstituting the G1 donor cells with the appropriate recipient cells. Recloning can be accomplished by separating the pre-implantation stage embryos into individual cells and then fusing each to the appropriate recipient cytoplast. As will be appreciated by those skilled in the art, such germ cell (or blastomere) cloning involves the cell cycle between donor and recipient cells to avoid chromosomal abnormalities and optimize development. Need to be adjusted. Preferably, the donor cells are obtained when the first generation cloned embryo is at the morula stage (approximately 32 cells in cattle), although the embryo may be at an earlier or later stage of development. Good. Alternatively, cloned fetuses generated from G1 cultured donor cells may be first developed, and then the fetal cell line may be re-derived and the embryo re-cloned using this new cell line. This recloning approach may provide benefits to nuclear reprogramming by increasing the time during which the initial donor nucleus is exposed to the oocyte cytoplasm during the initial pre-implantation period. This approach has the further advantage of increasing the number of cloned embryos obtained from first generation founder embryos to produce second generation, third generation etc. cloned embryos.
[0057]
Sequential nuclear transfer includes sequential nuclear transfer into the appropriate recipient cell cytoplasmic environment. In one embodiment of the present invention, it is desirable to first reconstitute a one-cell embryo using G1 donor cells and non-activated recipient cytoplasts with high MPF. This allows for NEBD and PCC, followed by the activation stimulus, which results in the formation of intact diploid nuclei. This nucleus (with nucleus remodeling and some nuclear reprogramming) is then aspirated from the one-cell cloned embryo as a nucleoplasm and then transferred to an enucleated fertilized egg at the appropriate stage after fertilization. can do. Thereafter, embryo development is allowed to proceed. Such a sequential serial nuclear transfer process could improve nuclear reprogramming. In addition, this may result in improved nuclear development in the second nuclear transfer, as a result of the introduction of nuclei into the cytoplasmic environment, which has undergone fertilization by sperm and is more appropriately activated for embryonic development.
[0058]
Production of transgenic animals
Generating a transgenic animal by nuclear transfer using a genetically modified donor nucleus at the G1 stage of the cell cycle involves pronuclear injection of DNA into fertilized eggs (Wall et al., 1997), or It is expected to be a more efficient and versatile approach than sperm-mediated transformation (Perry et al., 1999), which injects sperm-bound DNA into the cytoplasm. The advantages of this nuclear transfer technique using cultured cells are that (1) a much wider range of genetic manipulation is possible, and (2) cell lines, as opposed to collecting oocytes or fertilized eggs, (3) that all resulting clonal progeny are transgenic and of the desired gender, (4) pronuclear injection techniques, or sperm-mediated Includes the opportunity to speed up sheep and cattle herds that produce useful products in a shorter period of time than creating individual founder animals by transformation.
[0059]
Cultured cells can be genetically altered in any known manner to insert, remove, or modify the desired DNA sequence. Modifications include random insertion of a new DNA sequence (which can be heterologous), DNA insertion, deletion, or homologous recombination that allows for DNA insertion, deletion, or modification at specific sites in the genome. After selecting the cells and performing DNA analysis using methods well known in the art to confirm the desired genetic modification, karyotypically normal transgenic cells are selected in the G1 phase of the cell cycle and cloned / transformed. It can be used for nuclear transfer to produce transgenic animals.
[0060]
Depending on the particular gene being manipulated, there are diverse possibilities available for genetically modified livestock, both for biomedical and agricultural purposes. Potential fields include (1) the production of pharmaceutical proteins in milk, blood or urine, (2) the production of functional and medical foods, for example in milk, (3) the production of agricultural products. Manipulation of properties, such as improving the quantity and quality of milk, meat and fiber, improving disease and pest resistance, (4) production of industrial proteins in milk, for example, (5) xenograft, (6) human And the production of livestock as disease models for diseases such as cystic fibrosis and Huntington's disease.
[0061]
Therapeutic cloning
Significantly affected by nuclear transfer and embryonic stem cell technology may be the field of human cell-based therapy (Pedersen, 1999). Patients with certain diseases or injuries in tissues that cannot be effectively repaired or replaced by themselves (which occur in a variety of neurological disorders, including diabetes, muscular dystrophy, spinal cord injury, certain cancers, and Parkinson's disease) May be able to create their own therapeutic tissue and provide long-term or life-long treatment. First, this approach will use human nuclear transfer. This can include taking a small sample of healthy tissue from a human patient with a particular disease or injury, stimulating cell growth with media. By selecting donor cells in G1 of the cell cycle and fusing them to appropriate recipient cells, the nucleus could be reprogrammed. If the recipient cells are enucleated human oocytes, the reconstituted embryos will be able to grow to the blastocyst stage in a suitable embryo culture medium after a suitable activation stimulus. Then, under appropriate culture conditions (Thomson et al., 1998), human embryonic stem cells can be obtained from the inner cell mass of the cloned embryo, which is genetically consistent with the patient who received the cultured cells.
[0062]
By the term "embryonic stem cells", Applicants believe that cells isolated from any pluripotent cell type present inside the embryo, preferably at the blastocyst stage by methods well known in the art. A cell separated from the inner cell mass of an embryo.
[0063]
Embryonic stem cells produced by the methods of the present invention are undifferentiated, pluripotent (capable of differentiating into almost any cell type in the body), and have essentially unlimited proliferative potential in vitro. Alternatively, cell lines isolated from the inner cell mass may have differentiated to some extent and have a more limited ability to differentiate into diverse cell types, but will still be useful in therapy. Based on the experience of mouse embryonic stem cells, the appropriate conditions can be found, which can be used to treat specific diseases, such as neurons, hematopoietic cells, cardiomyocytes, etc. A population can be produced (eg, insulin-producing spleen cells for diabetes, or dopamine-producing neurons for Parkinson's disease). These genetically matched cells are then re-administered to a human patient to regenerate normal tissue at the site where they were implanted. Since the cells are genetically identical to the patient, they are not rejected, and therefore require little or no immunosuppressive drugs. Furthermore, after systematically modifying loci such as major histocompatibility complex genes that play an important role in the recognition of allogeneic cells by the immune system, "universal" embryonic stem cells for allogeneic transplantation It may also be beneficial to create a line.
[0064]
For some applications, genetic modification of embryonic stem cells may be required prior to differentiation and transplantation. This is for gene therapy, such as delivering therapeutic agents for treatment or correcting somatic genetic defects such as those occurring in the dystrophin gene in skeletal muscle of Duchenne muscular dystrophy patients.
[0065]
Differentiation of human embryonic stem cells into specific cell types will also be beneficial for drug discovery and drug toxicity studies in humans, in addition to transplantation therapy using cells, tissues, or organs.
[0066]
In addition, cells, tissues and organs can be isolated from the pups of a non-human cloned animal and used to transplant into a human patient in need of such treatment (xenograft). Where such cells, tissues, or organs contain a transgene, such cells, tissues, or organs may be useful in gene therapy or because they can suppress a patient's immune response to xenogeneic tissues. .
[0067]
Recipient cells for human applications
In the method of nuclear transfer according to the present invention, a preferred recipient is an enucleated oocyte prepared by the method described above. However, for human applications, this may be difficult.
[0068]
Alternatively, embryonic stem cells could be used as a source of recipient cells to reprogram differentiated somatic nuclei. Thus, somatic cells from a patient in need of any cell-based therapy can be cultured and dedifferentiated without the need for human oocytes. This can be achieved by fusing healthy somatic cells in G1 of the cell cycle with enucleated embryonic stem cells, or a group of embryonic stem cells (to provide more cytoplasm for reprogramming). it can. It is necessary to control the cell cycle status of the recipient stem cells, preferably in the M or G1 phase at the time of fusion. In this way, the differentiated nucleus of somatic cells will dedifferentiate after being exposed to the cytoplasm of stem cells. The resulting reconstituted cells are pluripotent or pluripotent and can be induced to form a series of differentiated cells useful for therapy. This concept has previously been demonstrated in hybrid cells created by fusing thymic lymphocytes with embryonic germ cells (Tada et al., 1997).
[0069]
The present invention will now be illustrated by the following examples, which do not limit the scope of the invention, as will be appreciated by those skilled in the art.
[0070]
Embodiment 1 FIG. Effect of follicular donor cells tuned to either G0 or G1 on in vitro development after nuclear transfer
In this particular experiment, the primary cell line of follicular granulosa cells was used for nuclear transfer. This cell line was designated "J1" and was derived from New Zealand Jersey heifers. J1 cells were used in this experiment between 7-8 subcultures.
[0071]
Donor cells were synchronized to two cell cycle stages for comparison.
(1) G0 cells were obtained by culturing for 10 to 11 days in a medium containing 0.5% FCS after serum collection.
(2) G1 cells were fused to cytoplasts within 1 to 3 hours after mitosis.
Negative BrdU labeling confirmed that J1 cells fused within 3 hours of mitosis were not in S phase and were in G1.
[0072]
In this experiment, embryos reconstituted with donor cells from two cell cycle treatment groups were cultured in vitro in AgR SOF medium supplemented with Life Technologies BSA (Life Technologies part number 30036-578). .
[0073]
Table 1 shows the results of in vitro generation. Donor cells in the G0 (control) and G1 phases of the cell cycle were equally efficient when fused with cytoplasts (recipient cells) by electro-mediated fusion. In addition, as shown in Table 1 below, there was no difference between the G0 and G1 donor cell-treated groups in the ratio of fused reconstituted embryos that developed into blastocysts after in vitro culture.
[0074]
Table 1: In vitro development of cloned embryos reconstituted with J1 follicle cells in either the G0 or G1 phase of the cell cycle and cultured in AgR SOF medium supplemented with Life Technologies BSA (mean ± standard error)
[Table 1]

[0075]
Embodiment 2. FIG. Effect of follicular donor cells (J1 and EFC cell lines) synchronized to either G0 or G1 on in vitro development cultured in medium supplemented with Sigma BSA after nuclear transfer
Further evidence supporting the finding that there is no effect of cell cycle stage between G0 and G1 follicular donor cells on the in vitro development of the cloned embryo into the blastocyst stage after 7 days of culture is provided in Table 2.
[0076]
The experiment was performed with two independent follicular cell lines.
(1) "EFC"; follicular cell line from New Zealand Friesian cows, and
(2) "J1": follicular cell line from New Zealand Jersey heifers.
In these experiments, EFC cells were used only in G0 of the cell cycle and J1 cells were used only in G1. However, the data were combined in this example since both cell lines were derived from follicular cells and the reconstituted embryos were all cultured in the same media formulation. In these experiments, this was a standard AgR SOF medium, but Sigma BSA (Sigma Chemical Company; product number A-7030) instead of Life Technologies BSA in Example 1 above. Supplemented.
[0077]
J1 cells were used for nuclear transfer between 3-6 passages with donor cells fused within 1-3 hours after mitosis. EFC cells were used between 3-8 passages and synchronized to G0 phase by culturing for 9-18 days in medium containing 0.5% serum.
[0078]
The in vitro development results shown in Table 2 show that in follicular donor cells, there is no difference in the proportion of fused embryos that develop into blastocysts between the G0 and G1 cell cycle stages.
[0079]
Table 2. In vitro development of cloned embryos reconstituted by follicular cells (J1 or EFC) in either G0 or G1 of cell cycle and cultured in AGR SOF medium supplemented with Sigma BSA (mean ± standard error)
[Table 2]

[0080]
Embodiment 3 FIG. Effect of follicular donor cells (J1 and EFC cell lines) synchronized to either G0 or G1 on in vivo development after nuclear transfer
The data shown in FIG. 1 shows the survival during pregnancy of the cloned bovine embryos reconstituted by follicular cells at either G0 or G1 of the cell cycle after introduction into recipient cows. The data in FIG. 1 is compiled from the experiments specifically described in Examples 1 and 2 above. This includes cloned embryos generated from two follicular cell lines, J1 and EFC. In addition, it includes embryos generated with the same AgR SOF media formulation, except supplemented with either Sigma BSA or Life Technologies BSA. There is no effect on viability of posttransfection between any of these two follicular cell lines, and no effect of the two sources of BSA (significant effect on blastocyst development) But) pooled the data.
[0081]
The data in FIG. 1 show that a total of 85 embryos reconstituted with G0 donor cells and a total of 95 embryos reconstituted with early G1 donor cells were introduced into the reproductive organs of synchronized recipient cattle. Represents an embryo.
[0082]
FIG. 1 plots the percentage of embryos / fetuses present throughout pregnancy from day 7 of embryo transfer to expected birth as measured by conventional ultrasonography, rectal examination and spontaneous delivery. Most notably, the use of G1 follicle cells has resulted in the birth of full and viable calves. Compared to G0 donor cells, the embryonic survival of cloned embryos reconstituted with G1 cells tended to be lower throughout the entire period from gestation day 30 to full birth, but this is not the case. Did not reach the statistical significance level with the number of introductions reported in.
[0083]
In G1 follicle cells, 9% (9/95) of the transferred embryos were full and resulted in calf birth. However, four of these calves died at birth or shortly after birth, resulting in an overall efficacy rate of viable cloned calves from G1 cells of 5% (5/95). In contrast, G0 donor follicle cells develop 20% (17/85) by the expected birth date, 3 calves die during calving, and 16% (14%) of viable calves as a whole. / 85).
[0084]
Embodiment 4. FIG. Effect of adult bovine skin fibroblasts (Age + and Age- cell lines) synchronized with G0 or G1 on in vitro and in vivo development after culture in medium supplemented with nuclear transfer and Life Technologies BSA
Data from experiments on two independent adult bovine skin fibroblast primary cell lines have been combined. The two cell lines are labeled "age +" and "age-". They are both female cell lines from Angus beef cattle, each selected for either late onset or early onset of the ripening period. Since there is no difference between these two analogous cell lines for in vitro and in vivo development, the data are combined in Table 3 below and FIG.
[0085]
Two cell cycle stages were compared in these experiments.
(1) G1: The donor cells were fused to the cytoplast within 1 to 3 hours after the end of mitosis.
(2) G0: Donor cells were cultured in a medium supplemented with 0.5% FCS for 3 to 12 days. For these reconstituted embryos introduced into recipient cows, serum was removed from the cells for 4-5 days.
[0086]
Cells from both cell lines and both cell cycle treatments were used for nuclear transfer in seven subcultures. Reconstituted embryos were cultured in a standard AgR SOF media formulation supplemented with Life Technologies BSA (Life Technologies product number 30036-578).
[0087]
The data presented in Table 3 show the effect on the fusion efficiency of the length of time that G0 cells have been in low serum, but once fused with the cytoplast had no effect on subsequent in vitro development. , G0 blastocyst data were combined. There was no effect of cell cycle stage between G0 and G1 on development to blastocyst stage.
[0088]
Table 3. In vitro development (average) of cloned bovine embryos reconstituted with adult female dermal fibroblasts (Age + and Age-) at G0 or G1 in the cell cycle and cultured in AgRSOF medium supplemented with Life Technologies BSA ± standard error)
[Table 3]

[0089]
The data in FIG. 2 show that the adult calf skin fibroblasts selected in G1 can fill the moon and produce viable calves. Similar to the data in FIG. 1, G0 has a greater tendency to survive on the expected birth date, but this is not statistically significant from the number of transfers performed here. For G1 donor cells, 4% (1/25) of the transferred embryos produced viable cloned cows at calving day. In contrast, GO donor cells produced 14% (3/22) viable development at birth date. In this experiment, all four calves survived after birth.
[0090]
Embodiment 5 FIG. Effect of adult bovine skin fibroblasts (LJ801 and 3XTC cell lines) synchronized with G0 or G1 on in vitro and in vivo development after nuclear transfer and culture in medium supplemented with ICP BSA
Additional experiments comparing the G0 and G1 cell cycle stages in two independent adult bovine skin fibroblast cell lines were performed. The two cell lines were labeled "LJ801" and "3XTC". LJ801 is a male cell line derived from a male calf of the Limosine X Jersey breed, and 3XTC is derived from a cross-bred calf that gave birth to three calves three times.
[0091]
Two cell cycle stages were compared:
(1) G0 cells in which the donor cells in both cell lines were cultured for 4-5 days in a medium with 0.5% FCS; and
(2) G1 cells in which donor cells have been fused to the cytoplast within 1 to 3 hours after mitosis.
[0092]
Negative BrdU labeling confirmed that the fused calf skin fibroblasts did not enter the S phase within 3 hours after mitosis.
Both LJ801 and 3XTC were used for nuclear transfer experiments in 3-4 subcultures.
[0093]
Embryos reconstituted from both cell lines with no difference in developmental efficiency to the blastocyst stage between LJ801 cells and 3XTC cells were now identified by ICP (Immunochemical Products, Auckland, New Zealand; product number ABFF-002) The data from Table 4 are combined, since they were cultured in the same standard AgRSOF medium supplemented with another source of BSA from ()). After embryo transfer, data on embryo survival through pregnancy are shown in FIGS. 3 and 4 for cell lines 3XTC and LJ801, respectively.
[0094]
The data in Table 4 show that there was no effect of the donor adult bovine skin fibroblast cell cycle synchronized to either G0 or G1 on blastocyst stage development. Table 4. In vitro development of cloned embryos reconstituted with adult bovine dermal fibroblasts (LJ801 and 3XTC cells) and cultured in AgR SOF medium supplemented with ICP BSA at either G0 or G1 of the cell cycle (mean ± standard error)
[Table 4]

[0095]
The data in FIG. 3 show that embryos reconstituted with 3XTC cells did not differ in survival by at least 150 days. Embryo survival at 150 days of donor cells in G1 of the cell cycle was 25% (3/12), compared to 27% (3/11) in G0 donor cells.
[0096]
The data in FIG. 4 shows that embryos reconstituted with LJ801 fibroblasts have a greater propensity to survive on day 210 of gestation with G0 donor cells, but this is not statistically significant. For donor cells at G1 of the cell cycle, embryo survival at 210 was 23% (3/13), whereas for GO donor cells was 39% (7/18).
[0097]
Embodiment 6 FIG. Effect of Genetically Altered Bovine Fetal Fibroblasts Synchronized with either G0 or G1 on In Vitro and In Vivo Development After Nuclear Transfer This experiment demonstrates the genetically altered fetal bovine female lung fibers Performed on blasts, the effect of cell cycle stage on development after nuclear transfer was examined. Genetic changes were made by random insertion of additional copies of the bovine β and κ-casein genes. This transgenic cell line was labeled "Casein Plus 5110".
[0098]
Two cell cycle stages were compared:
(1) G0 cells obtained by culturing donor cells in a medium having 0.5% FCS for 3 to 6 days; and
(2) G1 cells in which donor cells have been fused to the cytoplast within 1 to 3 hours after mitosis.
Reconstituted embryos were cultured in a standard AgR SOF medium composition supplemented with ICP BSP (Immunochemical Products, Auckland, New Zealand; product number ABFF-002).
[0099]
Table 5 shows data on in vitro embryo development. The development to the blastocyst stage by Casein Plus 5110 cells was significantly less when the donor cells used for nuclear transfer were in G1 than in G0. This result is in contrast to the other cell lines shown in the previous example.
[0100]
Table 5. Of cloned bovine embryos reconstituted with transgenic female lung dermal fibroblasts (Casein plus 5110 cells) at either the G0 or G1 phase of the cell cycle and cultured in AgR SOF medium supplemented with ICP BSA In vitro generation (mean ± standard error)
[Table 5]

[0101]
The embryo survival data shown in FIG. 5 shows that casein plus 5110 transgenic cells have no effect on cell cycle stage on development by at least 90 days. Embryo survival at day 90 was 38% (9/24) for donor cells at G1 of the cell cycle, while 27% (6/22) for G0 donor cells.
[0102]
Embodiment 7 FIG. Effect of non-proliferating senescent donor cells (in late G1 phase) on in vitro and in vivo development after nuclear transfer
Nuclear transfer experiments were performed to determine the effect on development by non-proliferating aged bovine donor cells. The cells used in this experiment were female fetal lung fibroblasts, which were genetically modified (designated as "561 cells"). Initially, the cells proliferate vigorously, but during the course of the cultivation, this gradually slows down, and during late passage, the cells enter a non-proliferative phase known as senescence by those of skill in the art, thereby causing cell differentiation. Stops. Senescent cells are known to be arrested at the G1 of the cell cycle, specifically at the late G1 / S phase boundary (Sherwood et al., 1988). When cells enter senescence, they are blocked in late G1 and do not enter S phase in response to physiological mitogens. Thus, aging is different from stationary phase. When the cells in the stationary phase are returned to the optimal conditions (for example, in the case of serum-deficient cell culture, additional serum), they are induced to re-enter the cell cycle and proliferate.
[0103]
Embryos reconstituted with the transgenic senescent cells were cultured in AgRSOF medium supplemented with ICP BSA.
Table 6 shows data on in vitro embryo development. The development to the blastocyst stage by senescent donor cells was lower than expected for the G0 or early G1 donor cells shown in the previous example.
[0104]
Table 5. In vitro generation of cloned bovine embryos reconstituted with non-proliferating senescent transgenic fibroblasts (561 cells) and cultured in AgR SOF medium supplemented with ICP BSA
[Table 6]

[0105]
The embryo survival data shown in FIG. 6 shows that cloning with non-proliferating senescent cells arrested in late G1 phase results in low efficiency with only 4% (1/26) of embryos developing by day 90. Implies that
[0106]
(Conclusion)
1. It is possible to select individual cells at certain stages of the cell cycle, preferably early G1. This is an advantage compared to the prior art using so-called proliferating cells, where the actual cell cycle stage in the individual cells used for nuclear transfer is not exactly known. The same is true for serum starved cell populations.
[0107]
2. Post-mitotic cells in the early G1 phase of the cell cycle are totipotent after nuclear transfer, as evidenced by live birth calves.
3. Thus, G0 is not the only cell cycle stage that is compatible with development after nuclear transfer using differentiated cultured cells, and nuclei can be functionally reprogrammed even at G1, preferably early G1.
[0108]
4. Early post-mitotic G1 cells promote development to the blastocyst stage after nuclear transfer to a similar level as cells in G0.
5. There is no significant difference in survival after transfer of cloned embryos reconstituted with either G0 or early G1 donor nuclei by the expected birth date.
[0109]
(Industrial applicability)
The present invention is particularly applicable to humans in the field of therapeutic cloning, as well as the cloning of animals such as transgenic animals capable of producing agriculturally useful products such as pharmaceutically useful proteins, meat, milk, fiber and the like. Useful for establishing groups.
[0110]
The description herein is not intended to limit the scope of the invention only to the above examples, but many modifications readily occurable to those skilled in the art without departing from the scope of the appended claims. It will be appreciated that this is possible.
[0111]
(References)
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[Brief description of the drawings]
FIG.
FIG. 3 shows the survival rate during pregnancy of cloned bovine embryos reconstituted using follicle cells with a donor cell cycle of either G0 or G1.
FIG. 2
FIG. 3 shows the viability during pregnancy of cloned bovine embryos reconstituted using adult female dermal fibroblasts with a donor cell cycle in either G0 or G1.
FIG. 3
FIG. 2 shows the viability during gestation of cloned bovine embryos reconstituted using adult female dermal fibroblasts (3XTC cells) with a donor cell cycle in either G0 or G1.
FIG. 4
FIG. 2 shows embryo survival during gestation of cloned bovine embryos reconstituted with adult male dermal fibroblasts (LJ801 cells) with donor cell cycle at either G0 or G1.
FIG. 5
Embryo survival during gestation of cloned transgenic bovine embryos reconstituted with genetically modified female fetal lung fibroblasts (casein plus 5110 cells) with donor cell cycle G0 or G1 FIG.
FIG. 6
FIG. 2 shows embryo survival during pregnancy of cloned transgenic bovine embryos reconstituted using non-proliferating senescent female fetal fibroblasts (561 cell line).

Claims (55)

A method of nuclear transfer comprising selecting and separating G1 cells from a growing or non-proliferating population of donor cells, and transplanting nuclei obtained from such separated G1 cells into enucleated recipient cells. 2. The method of claim 1, wherein the donor cell population is at one or more known or unknown stages of the cell cycle. 3. The method of claim 1 or 2, wherein the donor cell population is non-proliferating and is synchronized at any time during the G1 phase of the cell cycle. 4. The method according to any one of claims 1 to 3, wherein the G1 cells have been separated in the early G1 phase. 4. The method of any one of claims 1 to 3, wherein the donor cell population is non-proliferating and consists of senescent cells. 6. The method according to any one of claims 1 to 5, wherein the donor cell population is derived from an embryonic, fetal, juvenile, or adult cell isolated from an animal in vivo or from a cell culture in vitro. The described method. 7. The method of claim 6, wherein the donor cell population consists of any diploid karyotypic normal cells that can be stimulated to enter the cell cycle and proliferate. 8. The method of claim 7, wherein the donor cell population is in an undifferentiated cell state or at any stage of differentiation or at a quiescent or senescent stage. The method according to any of the preceding claims, wherein the donor cells are adult or fetal fibroblasts or follicles. The method according to any of the preceding claims, wherein said donor cells comprise modified cells. 11. The method of claim 10, wherein said donor cells comprise transgenic cells. The method of any of the preceding claims, wherein the recipient cells comprise enucleated oocytes. 13. The method of claim 12, wherein the enucleated oocyte is obtained from a species that is consistent with the donor nucleus. 12. The method according to any one of the preceding claims, wherein the recipient cells consist of clumps of enucleated stem cells or enucleated fused stem cells. 15. The method of claim 14, wherein the stem cells are embryonic stem cells isolated from a growing embryo or form an established cell line in culture. A method for producing a cloned animal embryo, comprising transferring an isolated donor nucleus at the G1 stage of the cell cycle into an enucleated recipient cell. 17. The method of claim 16, wherein the donor nucleus is genetically modified using methods well known in the art for producing cloned embryos having the desired genetic characteristics. 18. The method according to claim 16 or 17, wherein a cloned embryo of an animal species selected from the group consisting of birds, amphibians, fish and mammals is produced. 19. The clonal animal embryo is of a mammal selected from the group consisting of primates, including humans, rodents, rabbits, cats, dogs, horses, cows, sheep, deer, goats and pigs. The method described in. A reconstituted animal embryo prepared by the method of claim 16. 18. The reconstituted animal embryo of claim 17, comprising a transgenic embryo. 22. The reconstituted animal embryo according to claim 20 or 21, which is recloned to further increase embryo numbers or undergoes a series of nuclear transfers to promote nuclear reprogramming and / or development. 23. Any one of claims 20 to 22, comprising one of the mammals selected from the group consisting of primates, including humans, rodents, rabbits, cats, dogs, horses, cows, sheep, deer, goats and pigs. Item 10. The reconstituted animal embryo according to item 1. (1) producing a non-human cloned animal embryo by the method of any one of claims 16 and 17; (2) generating a non-human animal from the embryo at a predetermined time by a known method. And (3) a method of cloning a non-human animal, comprising the step of optionally breeding from a non-human animal formed by either conventional methods or additional cloning. The method according to claim 24, wherein the non-human cloned animal is a non-human mammal selected from the group consisting of non-human primates, rodents, rabbits, cats, dogs, horses, cows, sheep and deer. The described method. 26. The method of claim 24 or 25, wherein said non-human cloned animal is a non-human transgenic animal having the desired genetic characteristics. 27. The method of claim 26, wherein said non-human transgenic animal is a transgenic cow or sheep. A non-human cloned animal prepared by the method of claim 24. 29. The non-human cloned animal of claim 28, which is a mammal selected from the group consisting of a non-human primate, rodent, rabbit, cat, dog, horse, cow, sheep and deer. 30. The non-human cloned animal of claim 28 or 29, which is a non-human transgenic animal having the desired genetic characteristics. 31. The non-human cloned animal of claim 30, which is a transgenic cow or sheep. The desired genetic trait is the production of a pharmaceutical protein in milk, blood or urine; the production of a functional product in milk or meat; the beneficial produce traits to improve the quality of milk, meat and fibrous production. Production; improvement of resistance to pests and diseases; production of industrial proteins in milk; xenotransplantation; and insertion, deletion or modification of one or more genes for the creation of transgenic animals as human disease models 32. The transgenic cloned animal according to claim 30, which is selected from the group consisting of: 33. Offspring and progeny of a non-human cloned animal according to any one of claims 28 to 32. a) G1 cells are selected from a growing population of donor cells, a synchronized population of G1 cells, or a population of senescent cells, separated, and transplanted into enucleated recipient cells with nuclei obtained from the separated cells. B) growing to the blastocyst stage, c) harvesting the embryonic cells, and d) establishing an infinite dividing cell line in vitro by methods known in the art. A method for producing an embryonic cell line. 35. The method of claim 34, wherein said embryonic cells are embryonic stem cells. 36. The method of claim 34 or 35, wherein said donor cells are human cells. 37. The method according to any one of claims 34 to 36, wherein the donor and recipient cells are both human cells. 38. The donor cell is an adult or fetal cell selected from a normal karyotype, and the recipient cell is selected from any cell type capable of reprogramming gene expression. The method according to any one of claims 1 to 4. An embryonic cell line produced by the method of any one of claims 34 to 36. 37. A human embryonic stem cell line produced by the method of claim 36, dependent on claim 35, useful for therapeutic use. a) G1 cells are selected from a growing population of donor cells, or a synchronized population of G1 cells, or a population of senescent cells, isolated, and transplanted into enucleated recipient cells with nuclei obtained from the separated cells. A method of producing embryonic stem cells, comprising the steps of: (b) growing the cells at the blastocyst stage; and (c) collecting the embryonic stem cells. 42. The method of claim 41, wherein said donor cells are human cells. 43. The method of claim 41 or 42, wherein the donor and recipient cells are both human cells. 42. The donor cell is an adult or fetal cell selected from any normal karyotype and the recipient cell is selected from any cell type capable of reprogramming gene expression. 44. The method of any one of claims 43. An embryonic stem cell produced by the method according to any one of claims 41 to 43. 46. The embryonic stem cell of claim 45, comprising a human embryonic stem cell. Differentiated types of cells or tissues selected from the group consisting of nerve cells, muscle cells, heart cells, hepatocytes, lung cells, kidney cells or any other cell type of interest can be prepared by methods well known in the art. The use of an embryo cell according to any one of claims 39, 40 and 45, wherein the embryo cell has been cultured using the embryo cell. 48. The use according to claim 47, wherein the embryonic cells are human embryonic stem cells according to claim 40 or 46. An embryonic stem cell is made according to any one of claims 35 and 41 to 43 from a donor cell derived from a subject and differentiated cells or tissue for transplantation into said subject or another subject in need of treatment Therapeutic cloning methods that are cultured to produce a cloning product. 50. The method of claim 49, wherein said embryonic stem cells comprise one or more transgenes to confer a desired genetic property in a productive differentiated cell used for transplantation. A method of treating a disease, disorder or injury that can be treated by transplantation of the differentiated cells or tissues, wherein a therapeutically effective amount of the differentiated cells or tissues produced by the method of claim 49 or 50 is required. The above method comprising applying to the patient. Such diseases, disorders or injuries may include various neurological disorders (eg, Parkinson's disease), diabetes, heart disease, muscular dystrophy, various hereditary diseases, certain cancers (eg, leukemia), spinal cord injuries, burns and other illnesses. The method according to claim 49 or 50, wherein the method is selected from: 48. A method for drug discovery or drug toxicity testing using in vitro differentiated human embryonic stem cells produced by the method of claim 47. 33. A method of xenotransplantation wherein cells, tissues and organs are isolated from the non-human cloned animal of any one of claims 28 to 32 and used to transplant into a human patient in need thereof. 32. The method of gene therapy wherein cells, tissues and organs contain a transgene and are isolated from a cloned non-human animal according to claim 30 or 31.

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