JP2000508177A - 人工染色体、該染色体の使用および人工染色体の製造方法 - Google Patents
人工染色体、該染色体の使用および人工染色体の製造方法Info
- Publication number
- JP2000508177A JP2000508177A JP9538116A JP53811697A JP2000508177A JP 2000508177 A JP2000508177 A JP 2000508177A JP 9538116 A JP9538116 A JP 9538116A JP 53811697 A JP53811697 A JP 53811697A JP 2000508177 A JP2000508177 A JP 2000508177A
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Abstract
Description
Claims (1)
- 【特許請求の範囲】 1.選択可能マーカーを含むDNAフラグメントを細胞に導入する段階と、 前記DNAフラグメントをゲノムDNAに取込んだ細胞が産生されるような選 択条件下で細胞を増殖させる段階と、 サテライト人工染色体〔SATAC〕を含む細胞を選択する段階とから成る人 工染色体の製造方法。 2.細胞の染色体の増幅可能領域の内部またはその近傍にDNAフラグメントを 導入することを特徴とする請求項1に記載の方法。 3.増幅可能領域がrDNAから成ることを特徴とする請求項2に記載の方法。 4.増幅可能領域が異質染色質から成ることを特徴とする請求項2に記載の方法 。 5.細胞の染色体の挟動原体異質染色質にDNAを導入することを特徴とする請 求項1または2に記載の方法。 6.細胞が哺乳類細胞であることを特徴とする請求項1に記載の方法。 7.更に、サテライト人工染色体を単離する段階を含むことを特徴とする請求項 1または2に記載の方法。 8.DNAフラグメントが、当該フラグメントを染色体の異質染色質領域にター ゲットするヌクレオチドの配列を含むことを特徴とする請求項1から7のいずれ か一項の方法。 9.ターゲッティングヌクレオチド配列がサテライトDNAから成ることを特徴 とする請求項8に記載の方法。 10.細胞がヒト細胞であることを特徴とする請求項1から9のいずれか一項に 記載の方法。 11.細胞が、魚類、昆虫類、爬虫類、両棲類、蜘蛛類または齧歯類の細胞であ ることを特徴とする請求項1から5及び7から9のいずれか一項に記載の方法。 12.請求項1から11のいずれか一項に記載の方法によって製造されるサテラ イト人工染色体。 13.実質的に純粋な単離されたサテライト人工染色体。 14.約50〜約450メガ塩基〔Mb〕から成るメガ染色体であることを特徴 とする請求項13に記載のサテライト人工染色体。 15.約250〜約400Mbから成ることを特徴とする請求 項13に記載のサテライト人工染色体。 16.約150〜約200Mbから成ることを特徴とする請求項13に記載のサ テライト人工染色体。 17.約90〜約120Mbから成ることを特徴とする請求項13に記載のサテ ライト人工染色体。 18.約15〜約60Mbから成ることを特徴とする請求項13に記載のサテラ イト人工染色体。 19.請求項1から11のいずれか一項に記載の方法によって製造される人工染 色体を含む細胞。 20.請求項12から19のいずれか一項に記載のサテライト人工染色体を含む 細胞。 21.哺乳類細胞であることを特徴とする請求項19または20に記載の細胞。 22.サテライト人工染色体がメガ染色体であり、方法が更に、 断片化用ベクターを導入し、これによって細胞内のメガ染色体のサイズを小さ くする段階と、 約15〜約60Mbのサテライト人工染色体を含む細胞を同定する段階とを含 むことを特徴とする請求項1から11のいずれか一項に記載の方法。 23.サテライト人工染色体がメガ染色体であり、方法が、 欠失形メガ染色体を含む細胞が産生される条件を細胞に作用させる段階を更に 含むことを特徴とする請求項1から11のいずれか一項に記載の方法。 24.前記条件が、X線照射と、染色体内部の塩基対合を不安定化する物質の存 在下の増殖とから選択されることを特徴とする請求項23に記載の方法。 25.前記物質がブロモデオキシウリジンであることを特徴とする請求項24に 記載の方法。 26.更に、約15〜約60Mbから成るサテライト人工染色体を含んでいる細 胞を選択する段階を含むことを特徴とする請求項22から25のいずれか一項に 記載の方法。 27.請求項22から25のいずれか一項に記載の方法によって製造された人工 染色体を含む細胞。 28.人工染色体が約10〜約60Mbから成るサテライト人工染色体であるこ とを特徴とする請求項19から21及び25から27のいずれか一項に記載の細 胞。 29.約10〜約60Mbから成ることを特徴とする請求項13に記載の実質的 に純粋な単離されたサテライト人工染色体。 30.細胞からサテライト人工染色体を単離する段階を更に含むことを特徴とす る請求項1から11及び22から26のいずれか一項に記載の方法。 31.単離が、 中期染色体を単離し、 内因性染色体からサテライト人工染色体を識別し、 内因性染色体からサテライト人工染色体を分離することによって行われること を特徴とする請求項30に記載の方法。 32.DNA配列特異的染料で染色体を染色することによってサテライト人工染 色体を内因性染色体から識別し、フローセルソーターによって分離することを特 徴とする請求項31に記載の方法。 33.選択可能マーカーを含むDNAフラグメントを細胞に導入する段階と、 前記DNAフラグメントをそのゲノムDNAに取込んだ細胞が産生されるよう な選択条件下で細胞を増殖させる段階と、 増殖した細胞から新規な動原体を含む細胞を選択する段階と、から成る人工染 色体の製造方法。 34.更に、新規な動原体を含む染色体を有している細胞を単 離する段階と、ソーセージ型染色体を有している細胞が産生される条件下で細胞 を増殖させる段階とを含むことを特徴とする請求項33に記載の方法。 35.更に、ソーセージ型染色体を有している細胞を単離する段階と、第一のサ テライト人工染色体が製造される条件下で細胞を増殖させる段階とを含むことを 特徴とする請求項34に記載の方法。 36.細胞内の染色体の増幅可能領域の内部またはその近傍にDNAフラグメン トを導入することを特徴とする請求項35に記載の方法。 37.増幅可能領域がrDNAから成ることを特徴とする請求項36に記載の方 法。 38.増幅可能領域が異質染色質から成ることを特徴とする請求項36に記載の 方法。 39.DNAを細胞の染色体の挟動原体異質染色質に導入することを特徴とする 請求項35または36に記載の方法。 40.更に、 第一サテライト人工染色体にターゲットされる断片化用ベタターを導入する段 階と、細胞を増殖させる段階と、第一サテラ イト人工染色体よりも小さい第二サテライト人工染色体を含む細胞を選択する段 階とを含むことを特徴とする請求項33から39のいずれか一項に記載の方法。 41.選択された細胞が、新規な動原体を含む二動原体型染色体を有しているこ とを特徴とする請求項40に記載の方法。 42.選択された細胞が、元二動原体型染色体と、新規な動原体を含むミニ染色 体とを有していることを特徴とする請求項40に記載の方法。 43.選択された細胞が、元二動原体型染色体を有していることを特徴とする請 求項40に記載の方法。 44.選択可能マーカーを含むDNAフラグメントを細胞に導入する段階と、 前記DNAフラグメントをそのゲノムDNAに取込んだ細胞が産生されるよう な選択条件下で細胞を増殖させる段階と、 増殖した細胞から二動原体型染色体を産生した細胞を選択する段階と、 異質染色質アームをもつ染色体を含んでいる細胞が産生されるような選択条件 下で細胞を増殖させる段階とから成る人工染色体の製造方法。 45.更に、異質染色質アームをもつ染色体を含んでいる細胞を選択する段階と 、染色体を不安定化する物質の存在下で前記細胞を増殖させる段階とを含むこと を特徴とする請求項44に記載の方法。 46.更に、約50〜約400Mbの異質染色質性の染色体を有している細胞を 同定する段階を含むことを特徴とする請求項45に記載の方法。 47.細胞内の染色体の増幅可能領域の内部またはその近傍にDNAフラグメン トを導入することを特徴とする請求項44から46のいずれか一項に記載の方法 。 48.増幅可能領域がrDNAから成ることを特徴とする請求項47に記載の方 法。 49.増幅可能領域が異質染色質から成ることを特徴とする請求項47に記載の 方法。 50.DNAを細胞の染色体の挟動原体異質染色質に導入することを特徴とする 請求項47に記載の方法。 51.サテライト人工染色体を胚細胞に導入する段階から成る(ヒト以外の)ト ランスジェニック動物の産生方法。 52.胚細胞が幹細胞であることを特徴とする請求項51に記 載の方法。 53.胚細胞が胚に存在することを特徴とする請求項51に記載の方法。 54.サテライト人工染色体が遺伝子産物をコートするヘテロロガスDNAであ ることを特徴とする請求項51から53のいずれか一項に記載の方法。 55.サテライト人工染色体が治療用物質をコードするヘテロロガスDNAを含 むことを特徴とする請求項51から54のいずれか一項に記載の方法。 56.抗HIVリホザイムが抗gagリボザイムであり、腫瘍抑制遺伝子がp5 3であることを特徴とする請求項55に記載の方法。 57.産生物が、発現によって(ヒト以外の)トランスジェニツク動物中の病原 体に対する免疫防御応答を誘発する抗原を含むことを特徴とする請求項54に記 載の方法。 58.産生物が、発現によって複数の病原体に対する免疫防御応答を誘発する複 数の抗原を含むことを特徴とする請求項54に記載の方法。 59.(ヒト以外の)トランスジェニック動物が魚類、昆虫類、 爬虫類、両棲類、蜘蛛類または哺乳類であることを特徴とする請求項51から5 8のいずれか一項に記載の方法。 60.サテライト人工染色体が、細胞融合、キャリア系による脂質仲介トランス フェクション、マイクロインジェクション、マイクロセル融合、エレクトロポレ ーション、マイクロプロジェクティル、核移入、衝撃または直接DNA移入によ って導入されることを特徴とする請求項51から59のいずれか一項に記載の方 法。 61.請求項51から60のいずれか一項に記載の方法によって産生される(ヒ ト以外の)トランスジェニック動物。 62.魚類、昆虫類、爬虫類、両棲類、蜘蛛類または哺乳類であることを特徴と するトランスジェニック動物。 63.選択可能マーカーを含むDNAフラグメントを細胞に導入する段階と、 前記DNAフラグメントをそのゲノムDNAに取込んだ細胞が産生されるよう な選択条件下で細胞を増殖させる段階と、 選択可能マーカーと真正染色質とを含む約10Mb〜約50Mbのミニ染色体 を有している細胞を選択する段階と、 ミニ染色体を単離して植物または動物の細胞に導入する段階 とから成るトランスジェニック植物または動物の産生方法。 64.細胞の選択後に、1つまたは複数の遺伝子産物をコードするDNAを細胞 に導入し、(1つまたは複数の)遺伝子産物をコードするDNAを含むミニ染色 体を有している細胞が産生される選択条件下で細胞を増殖させることを特徴とす る請求項63に記載の方法。 65.細胞の選択後に、1つまたは複数の遺伝子産物をコードするDNAを細胞 に導入し、(1つまたは複数の)遺伝子産物をコードするDNAを含むサテライ ト人工染色体を有している細胞が産生される選択条件下で細胞を増殖させること を特徴とする請求項64に記載の方法。 66.動物または植物の動原体のクローニング方法であって、 植物または動物のゲノムを含むDNAフラグメントのライブラリーを調製する 段階と、 選択された植物または動物の異なる種に由来の動原体と選択可能マーカーとを 各々が含む哺乳類サテライト人工染色体に前記フラグメントの各々を導入する段 階と、 前記サテライト人工染色体の各々を細胞内に導入して選択条件下で細胞を増殖 させる段階と、 サテライト人工染色体を有している細胞を同定する段階と、 これらの細胞から出発サテライト人工染色体の動原体とは異なる動原体を含む サテライト人工染色体を有している細胞を選択する段階とから成る方法。 67.それぞれ受託番号96040926、96040927、9604092 9及び96040928でECACCに寄託されたTF1004G19C5、1 9C5xHa4、H1D3及びG3D5のいずれかの同定形質を有する細胞系。 68.約50〜400Mbから成るメガ染色体を含む細胞系。 69.メカ染色体が250〜約400Mbから成ることを特徴とする請求項68 に記載の細胞系。 70.メガ染色体が約150〜約200Mbから成ることを特徴とする請求項6 8に記載の細胞系。 71.メガ染色体が約90〜約120Mbから成ることを特徴とする請求項68 に記載の細胞系。 72.メガ染色体が約60〜約100Mbから成ることを特徴とする請求項68 に記載の細胞系。 73.治療用物質のDNAを含むサテライト人工染色体をターゲット細胞に導入 する段階と、 得られたターゲット細胞を宿主動物に導入する段階とから成る遺伝子治療方法 。 74.ターゲット細胞が、リンパ球、幹細胞、神経細胞、昆虫細胞、ニワトリ細 胞または筋肉細胞であることを特徴とする請求項73に記載の方法。 75.ミニクロモソームがEC3/7C5細胞系に存在するミニ染色体であるこ とを特徴とする請求項73に記載の方法。 76.染色体がKE1 2/4細胞系に存在するλ neo染色体であることを 特徴とする請求項73に記載の方法。 77.約20Mb〜約200Mbであることを特徴とする請求項76に記載の人 工染色体。 78.約100Mb〜約200Mbであることを特徴とする請求項76に記載の 人工染色体。 79.約20Mb〜約200Mbであることを特徴とする請求項76に記載の人 工染色体。 80.約1Mb〜約15Mbであることを特徴とする請求項76に記載の人工染 色体。 81.動物が哺乳動物または卵生動物であり、サテライト人工染色体がタンパク 質と、動物の母乳または動物の卵の内部で遺 伝子を発現させる調節要素とを含むことを特徴とする請求項51に記載の方法。 82.動物が、雌ウシ、ヤギ、雄ウシ、ブタ及びヒツジから選択されることを特 徴とする請求項81に記載の方法。 83.動物が家禽から選択されることを特徴とする請求項81に記載の方法。 84.サテライト人工染色体がヒト細胞表面タンパク質発現用遺伝子をコードす るDNAを含み、それにより動物の器官がヒトタンパク質を発現し、ヒトに移植 されたときに拒絶が生じないことを特徴とする請求項51に記載の方法。 85.配列13、14または15で示される配列を有するDNAから成る単離D NA。 86.配列13、14または15で示される配列を有するDNAから成る単離D NA。 87.配列18から24のいずれかで示されるヌクレオチド配列を有する単離D NAフラグメント。 88.配列18から24のいずれかで示されるヌクレオチド配列を有することを 特徴とする請求項14に記載のサテライト人工染色体。 89.配列18から24のいずれかで示されるヌクレオチド配列を有することを 特徴とする請求項13に記載のサテライト人工染色体。 90.配列18から24のいずれかで示されるヌクレオチド配列を有することを 特徴とする請求項13に記載のサテライト人工染色体。 91.ヘテロロガス遺伝子の多数コピーまたは複数のヘテロロガス遺伝子から成 る人工染色体〔AC〕を含んでいる細胞から成る細胞性産生系。 92.人工染色体がサテライト人工染色体であることを特徴とする請求項91に 記載の細胞性産生系。 93.ヘテロロガス遺伝子が代謝経路を構成するタンパク質をコードすることを 特徴とする請求項91または92に記載の系。 94.代謝経路の発現によって産生される物質の発現方法であって、代謝経路を 構成するタンパク質が発現されて前記物質を産生する条件下で請求項93に記載 の系を培養する段階から成る方法。 95.産生物がビタミン、ホルモン、ヌクレオチド、アミノ酸、タンパク質また はペプチドであることを特徴とする請求項94 に記載の方法。 96.動物が卵生であることを特徴とする請求項51に記載の方法。 97.動物がニワトリであることを特徴とする請求項51に記載の方法。 98.動物が昆虫であることを特徴とする請求項51に記載の方法。 99.請求項13から18または88から90のいずれか一項に記載のサテライ ト人工染色体を植物細胞に導入する段階と、植物が発生する条件下で細胞を培養 する段階とから成るトランスジェニック植物の産生方法。 100.プロトプラスト融合、マイクロインジェクション、マイクロセル融合、 脂質仲介遺伝子導入、エレクトロポレーション、微粒子衝撃、または、直接DN A導入によってサテライト人工染色体を導入することを特徴とする請求項99に 記載の方法。 101.請求項13から18または88から90のいずれか一項に記載のサテラ イト人工染色体を細胞に導入する段階と、(1つまたは複数の)遺伝子産物が発 現される条件下で細胞を培養 する段階とから成る、(1つまたは複数の)遺伝子産物の産生方法。 102.代謝経路を構成するタンパク質をコードする一連の遺伝子の発現によっ て遺伝子産物が産生されること、及び、サテライト人工染色体がこれらの遺伝子 の各々を含むことを特徴とする請求項102に記載の方法。 103.動原体とテロメアとメガレプリケーターと選択可能マーカーとから成り 、動原体が請求項12から18及び88から90のいずれか一項に記載のサテラ イト人工染色体に由来することを特徴とするin vitro合成された哺乳類 人工染色体〔ISMAC〕。 104.動原体とテロメアとメガレプリケーターと選択可能マーカーとから成り 、動原体が請求項12から18及び88から90のいずれか一項に記載のサテラ イト人工染色体に由来することを特徴とするin vitro合成された哺乳類 人工染色体〔ISMAC〕。 105.更に、異質染色質を含むことを特徴とする請求項103または104に 記載のISMAC。 106.メガレプリケーターがrDNAから成ることを特徴と する請求項103から105のいずれか一項に記載のISMAC。 107.動原体がヒト動原体であることを特徴とする請求項103から106の いずれか一項に記載のISMAC。 108.動原体がメガ染色体に由来することを特徴とする請求項103から10 7のいずれか一項に記載のISMAC。 109.動原体がヨーロッピアン・コレクション・オブ・アニマル・セル・カル チャー・(ECACC)に受託番号96040929で寄託された細胞系の同定 形質の全部を有する細胞系に由来することを特徴とする請求項103、105、 106または108に記載のISMAC。 110.産生物がホルモン、抗体、サイトカイン、成長因子、調節タンパク質、 分泌タンパク質であることを特徴とする請求項54に記載の方法。 111.産生物が嚢胞性線維症トランスメンブラン調節タンパタ質(CFTR) 、抗HIVリボザイムまたは腫瘍抑制遺伝子であることを特徴とする請求項54 に記載の方法。 112.動物がヒトであることを特徴とする請求項66に記載の方法。 113.動原体とテロメアとメガレプリケーターと複製可能な ISMACと産生する選択可能マーカーとを組合せる段階から成り、動原体が請 求項12から18及び88から90のいずれか一項に記載のサテライト人工染色 体に由来することを特徴とするin vitro合成された哺乳類人工染色体〔 ISMAC〕の製造方法。 114.更に、ISMAC中にrDNAを含むことを特徴とする請求項113に 記載の方法。 115.テロメアが配列29の複数の反復を含むことを特徴とする請求項113 に記載の方法。 116.テロメアが約1kB〜約1Mbであり、好ましくは約1kB〜約500 kBであることを特徴とする請求項115に記載の方法。 117.テロメアが配列29の複数の反復を含むことを特徴とする請求項103 から108のいずれか一項に記載のISMAC。 118.テロメアが約1kB〜約1Mbであり、好ましくは約1kB〜約500 kBであることを特徴とする請求項117に記載のISMAC。 119.選択可能マーカーを含むDNAフラグメントを細胞に導入する段階と、 前記DNAフラグメントをそのゲノムDNAに取込んだ細胞が産生されるよう な選択条件下で細胞を増殖させる段階とから成り、 DNAフラグメントが細胞内の染色体の増幅可能領域の内部または近傍に導入 され、増幅可能領域に由来のDNAを含むミニ染色体が産生され、ミニ染色体が 異質染色質よりも多い真正染色質を含む人工染色体であることを特徴とする人工 染色体の製造方法。 120.増幅可能領域がrDNAであることを特徴とする請求項119に記載の 方法。 121.更に、ミニ染色体を単離する段階を含むことを特徴とする請求項119 または120に記載の方法。 122.請求項119から121のいずれか一項に記載の方法によって産生され るミニ染色体。 123.更に、遺伝子産物をコードするDNAを導入する段階を含み、遺伝子を コードするDNAが、選択可能マーカーを含むフラグメントに存在するかまたは 第二のDNAフラグメントに存在すること、及び、得られたサテライト人工染色 体が遺伝子産物をコードするヘテロロガスDNAを含むこどを特徴とする請求項 1に記載の方法。
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Cited By (10)
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US8288610B2 (en) | 1996-04-10 | 2012-10-16 | The Biological Research Center Of The Hungarian Academy Of Sciences | Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes |
US8389802B2 (en) | 1996-04-10 | 2013-03-05 | The Biological Research Center Of The Hungarian Academy Of Sciences | Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes |
JP2004508046A (ja) * | 2000-07-17 | 2004-03-18 | 夏,家輝 | ヒト由来の遺伝子先導配列、遺伝子ベクター及びその発現方法 |
JP2005500827A (ja) * | 2001-05-30 | 2005-01-13 | クロモス・モレキユラー・システムズ・インコーポレーテツド | 植物人工染色体、その使用及び植物人工染色体の製造方法 |
JP2009017884A (ja) * | 2001-05-30 | 2009-01-29 | Chromos Molecular Systems Inc | 染色体に基くプラットホーム |
JP2009254382A (ja) * | 2001-05-30 | 2009-11-05 | Calyx Bio-Ventures Inc | 植物人工染色体、その使用及び植物人工染色体の製造方法 |
JP4771656B2 (ja) * | 2001-05-30 | 2011-09-14 | カリックス・バイオ−ベンチャーズ・インコーポレイテッド | 植物人工染色体、その使用及び植物人工染色体の製造方法 |
JP2018504937A (ja) * | 2015-02-09 | 2018-02-22 | パーキンス、エドワードPERKINS, Edward | 哺乳動物合成染色体の構築をリアルタイムでモニタリングするための、及び哺乳動物合成染色体を生体工学的に操作するための組成物及び方法 |
JP2021072791A (ja) * | 2015-02-09 | 2021-05-13 | キャリージーンズ バイオエンジニアリングCarryGenes Bioengineering | 哺乳動物合成染色体の構築をリアルタイムでモニタリングするための、及び哺乳動物合成染色体を生体工学的に操作するための組成物及び方法 |
JP7089878B2 (ja) | 2015-02-09 | 2022-06-23 | キャリージーンズ バイオエンジニアリング | 哺乳動物合成染色体の構築をリアルタイムでモニタリングするための、及び哺乳動物合成染色体を生体工学的に操作するための組成物及び方法 |
Also Published As
Publication number | Publication date |
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NZ503055A (en) | 2002-07-26 |
US20070061920A1 (en) | 2007-03-15 |
HUP9901482A2 (hu) | 1999-07-28 |
EP0929689A2 (en) | 1999-07-21 |
JP2006000118A (ja) | 2006-01-05 |
EP0929689B1 (en) | 2013-08-28 |
JP4999293B2 (ja) | 2012-08-15 |
AU2451297A (en) | 1997-11-12 |
JP3754457B2 (ja) | 2006-03-15 |
US6025155A (en) | 2000-02-15 |
JP2008109947A (ja) | 2008-05-15 |
JP2004033209A (ja) | 2004-02-05 |
IL126126A (en) | 2010-05-31 |
IL126126A0 (en) | 1999-05-09 |
CA2250682C (en) | 2005-09-27 |
HUP9901482A3 (en) | 2001-06-28 |
CA2250682A1 (en) | 1997-10-30 |
WO1997040183A3 (en) | 1998-02-05 |
JP2004357716A (ja) | 2004-12-24 |
NZ331815A (en) | 2000-04-28 |
WO1997040183A2 (en) | 1997-10-30 |
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