JP4528940B2 - 高発現の宿主細胞を選択する方法 - Google Patents
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- C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
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Description
エンハンサーはリンクしたプロモータからの転写を刺激する。プロモーターとは異り、エンハンサーは、転写開始部位から下流に、またはプロモータから相当な距離で置かれた時、活性であるが、実際問題としてエンハンサーはプロモータと物理的および機能的にオーバーラップし得る。例えば、上に挙げた強力なプロモーターは強力なエンハンサーをも含む。Bendig、Genetic Engineering、7:91(Academic Press、1988)。
本明細書に開示する「DNA構築物」は、単離物として、または他のDNA分子中に、例えば発現ベクターまたは真核宿主細胞の染色体中に組み込まれて提供され得る、天然に存在しないDNA分子を含む。
駆体に導入された最初の核酸に対応するDNA構築物中の核酸を言い、その部位はDNA構築物の5'末端に、または5'末端に隣接して一般に設けられる。
写を増加させる、通常、約10〜300bpのシス作用性DNA因子)を通常言う。
発明の実施の形態
単一の機構が哺乳動物、植物および酵母細胞におけるmRNA前駆体のスプライシングに関与すると考えられる。一般に、プライシングの過程は、イントロンの5'および3'末端が正しく解裂し、生じたmRNAの末端が正確に結合することを必要とし、その結果タンパク質合成のための適当な読み枠を有する成熟mRNAが作られる。様々な、天然に存在する、および合成的に構築された変異体遺伝子の分析により、5'および3'スプライス部位におけるコンセンサス配列内部の場所の多くでのヌクレオチドの変化は成熟mRNAの合成を減少させ、または消滅させるという効果が示された。Sharp.Science、235:766(1987);Padgett等、Ann.Rev.Biochem.、55:1119(1986);Green、Ann.Rev.Genet.、20:671(1986)。変異の研究により、スプライシング部位を含むRNA2次構造はスプライシングの効率に影響することが示された。Solnick、Cell、43:667(1985);Konarska等、Cell、42:165(1985)。
或いは、遺伝子発現を、組織断片の免疫組織化学的染色、細胞培養物また体液の分析等の免疫学的方法により測定して、生成物遺伝子の発現を直接定量してもよい。免疫組織化学的染色技術の場合、細胞試料を典型的には脱水および固定により調製し、カップルする遺伝子生成物に特異的な標識した抗体と反応させる。標識は酵素標識、蛍光標識、発光標識等、通常視覚的に検知可能である。本発明に用いるのに適した特に感度の高い染色技術がHsu等、Am. J. Clin. Path.、75:734〜738(1980)により記載されている。
ジシストロン性発現ベクターを用いるtPAの製造
単一のプロモーターから選択可能なマーカー(DHFR等の)および問題のタンパク質を発現させることにより、安定なクローンの発現レベルに関して均一性のレベルを増加させることを探究した。
ジシストロン性発現ベクターを用いるTNFr−IgG製造
このアプローチの一般的な応用性を証明するために、問題のDNAとして、腫瘍壊死因子(TNF)を結合できるイムノアドヘシン(TNFr−IgG)(Ashkenazi等、Proc. Natl. Acad. Sci. U.S.A.、88:10535〜10539[1991])を含むジシストロン性ベクター系において第2の生成物を評価した。生成物遺伝子がイムノアドヘシンTNFr−IgGをコードすることを除いては、実施例1に記載の実験を本質的に繰返した。TNFr−IgG cDNA、および(a)WTras、すなわち図6(配列番号2)、(b)変異体rasまたは(c)非機能性スプライスドナー部位を含むプラスミドDNAを、実施例1について論じたようにdp12.CHO細胞中に導入した。DNA構築物の説明について図1Cを参照。
ジシストロン性発現ベクターを用いる抗体製造
抗体発現についての本システムの有用性を、IgEに対する抗体(Presta等、Journal of lmmunology、151:2623〜〜2632[1993])の製造を試験することにより評価した。さらに転写開始に関するシステムの柔軟性を、前のベクターに存在したCMVプロモーター/エンハンサーを、SV40ウイルスの初期領域に由来するプロモーター/エンハンサー(Griffin,B.、SV40およびポリオーマウイルスの構造およびゲノム組織、J.Tooze編、DNA Tumor Vivuses中、Cold Spring Harbor Laboratory、Cold Spring Harbor、ニューヨーク)で置換することにより試験した。該抗体の重鎖を、前のtPAおよびTNFr−IgG構築物に記載したようにDHFRの下流に挿入した。さらに、実施例1および2で試験したベクターに存在するスプライスドナー部位よりも、そのコンセンサススプライスドナー部位により密にマッチする、新しいスプライスドナー部位配列(GAC:GTAAGT)を、ベクター中に入れた。生じた発現ベクターを図1Dおよび9に示す。
本明細書に記載する有効な発現系は、プロモーター/エンハンサー因子、その後の選択可能なマーカーコード配列を有するイントロン、その後の問題のcDNAおよびポリアデニル化シグナルよりなるベクターを用いる。
/Lという力価を与えた。
Claims (17)
- 5'から3'の順に以下を含むDNA構築物:
(a)転写制御領域;
(b)転写開始部位;
(c)イントロンのスプライスドナー部位5'を有するイントロンの内部に位置する選択可能な遺伝子;その際、該スプライスドナー部位は該転写制御領域を用いて生成物遺伝子の発現を制御し、該選択可能な遺伝子は増幅可能な遺伝子である;
(d)目的生成物をコードする生成物遺伝子;および
(e)転写終了部位;
ここで、該転写制御領域は、該選択可能な遺伝子および該生成物遺伝子の両方の転写を制御する。 - スプライスドナー部位がコンセンサススプライスドナー配列を含有する請求項1に記載のDNA構築物。
- スプライスドナー部位が配列GACGTAAGTを含有する請求項1に記載のDNA構築物。
- 増幅可能な遺伝子がDHFRである請求項1〜3のいずれかに記載のDNA構築物。
- 転写制御領域がプロモーターおよびエンハンサーを含有する請求項1に記載のDNA構築物。
- 請求項1〜5のいずれかに記載のDNA構築物を含有するベクター。
- 真核宿主中で複製できる請求項6に記載のベクター。
- 請求項6または7に記載のベクターを含有する真核宿主細胞。
- 請求項1〜5のいずれかに記載のDNA構築物を含有する真核宿主細胞。
- 宿主細胞の染色体に組込まれた請求項1〜5のいずれかに記載のDNA構築物を含有する真核宿主細胞。
- 哺乳動物細胞である請求項10に記載の宿主細胞。
- 請求項8〜10のいずれかに記載の宿主細胞を培養して目的生成物遺伝子を発現させ、宿主細胞培養から生成物を回収することを含んでなる目的生成物の製造方法。
- 培地から生成物を回収することを含んでなる請求項12に記載の方法。
- 請求項8〜11のいずれかに記載の宿主細胞を培養して、十分な時間増幅剤を含有する選択培地中で生成物遺伝子を発現させて、増幅を起こさせ、生成物を回収することを含んでなる目的生成物の製造方法。
- 真核細胞を請求項1〜5のいずれかに記載のDNA構築物でトランスホームし、増幅剤を含む選択培地中で増幅が起こるのに十分な時間増殖させ、生成物遺伝子の多重コピーを有する細胞を選択することを含んでなる、生成物遺伝子の多重コピーを有する真核細胞の製造方法。
- DNA構築物を真核細胞にエレクトロポレーションにより導入する請求項15に記載の方法。
- 生成物遺伝子を発現させるよう選択した細胞を培養し、目的生成物を選択した細胞から回収することを更に含んでなる請求項15に記載の方法。
Applications Claiming Priority (1)
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US08/286,740 US5561053A (en) | 1994-08-05 | 1994-08-05 | Method for selecting high-expressing host cells |
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JP8506644A Division JPH10503376A (ja) | 1994-08-05 | 1995-07-28 | 高発現の宿主細胞を選択する方法 |
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JP2006296438A JP2006296438A (ja) | 2006-11-02 |
JP2006296438A5 JP2006296438A5 (ja) | 2008-10-23 |
JP4528940B2 true JP4528940B2 (ja) | 2010-08-25 |
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JP8506644A Withdrawn JPH10503376A (ja) | 1994-08-05 | 1995-07-28 | 高発現の宿主細胞を選択する方法 |
JP2006201808A Expired - Lifetime JP4528940B2 (ja) | 1994-08-05 | 2006-07-25 | 高発現の宿主細胞を選択する方法 |
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JP8506644A Withdrawn JPH10503376A (ja) | 1994-08-05 | 1995-07-28 | 高発現の宿主細胞を選択する方法 |
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US (1) | US5561053A (ja) |
EP (1) | EP0770136B1 (ja) |
JP (2) | JPH10503376A (ja) |
AT (1) | ATE353970T1 (ja) |
AU (1) | AU704408B2 (ja) |
CA (1) | CA2195303C (ja) |
DE (1) | DE69535389T2 (ja) |
DK (1) | DK0770136T3 (ja) |
ES (1) | ES2282994T3 (ja) |
IL (1) | IL114820A0 (ja) |
MX (1) | MX9700852A (ja) |
PT (1) | PT770136E (ja) |
WO (1) | WO1996004391A1 (ja) |
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JP4912144B2 (ja) | 2003-03-12 | 2012-04-11 | ジェネンテック, インコーポレイテッド | 造血促進のためのbv8及び/又はeg−vegfの使用 |
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US20060195935A1 (en) | 2004-11-08 | 2006-08-31 | Chromagenics B.V. | Selection of host cells expressing protein at high levels |
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DK0770136T3 (da) | 2007-06-11 |
EP0770136A1 (en) | 1997-05-02 |
IL114820A0 (en) | 1995-12-08 |
JPH10503376A (ja) | 1998-03-31 |
CA2195303A1 (en) | 1996-02-15 |
EP0770136B1 (en) | 2007-02-14 |
ES2282994T3 (es) | 2007-10-16 |
AU704408B2 (en) | 1999-04-22 |
CA2195303C (en) | 2008-01-15 |
US5561053A (en) | 1996-10-01 |
WO1996004391A1 (en) | 1996-02-15 |
AU3204595A (en) | 1996-03-04 |
DE69535389D1 (de) | 2007-03-29 |
DE69535389T2 (de) | 2007-10-31 |
MX9700852A (es) | 1997-04-30 |
ATE353970T1 (de) | 2007-03-15 |
JP2006296438A (ja) | 2006-11-02 |
PT770136E (pt) | 2007-05-31 |
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