JP2006509512A - 活性物質をスクリーニングするために遺伝学的に改変された生物を製造する方法 - Google Patents
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Abstract
Description
a)生物の遺伝学的改変によって、少なくとも1種のタンパク質またはタンパク質フラグメントの異種発現を起こす工程。
b)好ましくは、それに続いて、遺伝学的に改変された生物の表現型を決定する工程。
c)改変された遺伝子発現パターンを解析し、代償として(compensatingly)差異的に(differetially)調節された遺伝子を同定する工程。
d)前記生物を表現型解析する工程(好ましくは、代償として調節された遺伝子の欠失、変異誘発または過剰発現によって、異種発現されたタンパク質またはタンパク質フラグメントと協同して表現型を増強する、または発生させる)。
アーゼ、プロテアーゼおよびイオンチャンネルが、本発明の範囲内で特に好ましい。
実施例1:生物として酵母をベースとした、キナーゼの活性に作用する薬物を同定するためのプラットフォーム技術の開発
この場合において、生じる表現型は、酵母の成長阻害である。従って、本分析の原理は酵母の成長阻害に基づき、酵母は、生きている「試薬チューブ」として用いられる。ここで成長阻害とは、例えば、細胞周期の停止、または、関連する細胞の溶解を意味する。酵母は、遺伝学的な可操作性が理想的に適しているために用いられる。ヒト(またはその他の外因性)キナーゼは、酵母において、ガラクトース誘導性プロモーター(GAL1/10)の制御下で過剰発現される。酵母は、標準的な方法に従って形質転換され、培養される。用いられるベクターの例としては、p41x−GAL1、または、p42x−GAL11系のベクターが挙げられる。
ナーゼを代償として発現しなくなる。それゆえに、これらの系をHTSに送ることができる。
遺伝子操作については、Sambrook等(1989年)の、Molecular Cloning:A Laboratory Manual,第二版(コールドスプリングハーバーラボラトリープレス(Cold Spring Harbor Laboratory Press),コールドスプリングハーバー,ニューヨーク州,第545頁)による標準的な方法を用いた。
Brown, A.J.P. and M. Tuite (1998), PCR-Based Gene Targeting in Saccharomyces cerevisiae. Methods Microbiol. 26, 67-81.
Methods in Yeast Genetics; A Cold Spring Harbor Course Manual; 1994 Edition; Kaiser, C., Michaelis, S., and A. Mitchell; Cold Spring Harbor Laboratory Press.
Mumberg, D., Mueler, R. and M. Funk (1994). Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expresson. Nucl. Acids Res. 22, 5767-5768.
Tugendreich, S., Perkins, E., Couto, J., Barthmaier, P., Sun, D., Tang, S., Tulac, S., Nguyen, A., Yeh, E., Mays, A., Wallace, E., Lila, T., Shivak, D., Prichard, M., Andrejka, L., Kim. R. and T. Melese (2001). A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays. Genome Res. 11, 1899-1912.
Claims (22)
- 薬物スクリーニングのために、遺伝学的に改変された生物を製造する方法であって、
a)生物の遺伝学的改変によって、少なくとも1種のタンパク質またはタンパク質フラグメントの異種発現を起こす工程
b)改変された遺伝子発現パターンを解析すること、および、代償として差異的に調節された遺伝子を同定する工程
c)該生物を表現型解析する工程、
を含む、上記方法。 - 表現型解析が、代償としての差異的な発現を減少/除去させること、または、少なくとも1つの代償として差異的に調節された遺伝子を標識することによって行われる、請求項1に記載の方法。
- 遺伝学的改変が、生物にとって内因性および/または外来の少なくとも1種のタンパク質またはタンパク質フラグメントの異種発現を起こす、請求項1または2に記載の方法。
- 遺伝学的改変が、生物にとって内因性の少なくとも1種のタンパク質の発現の減少または除去を起こす、請求項1〜3のいずれか一項に記載の方法。
- 改変された発現が、誘導性である、請求項1〜4のいずれか一項に記載の方法。
- 遺伝学的改変が、タンパク質またはタンパク質フラグメントを誘導によって発現できるベクター、好ましくは、ガラクトース、銅テトラサイクリン誘導性ベクター、または、その他の同等の誘導性ベクターを導入することを含む、請求項5に記載の方法。
- 遺伝学的改変が、ノックアウト、好ましくは誘導性ノックアウトを含む、請求項1〜6のいずれか一項に記載の方法。
- 生物が、ショウジョウバエ、C.エレガンス、原核細胞または真核細胞である、請求項1〜7のいずれか一項に記載の方法。
- 細胞が、酵母細胞、好ましくはS.セレビジエ株の酵母細胞である、請求項8に記載の方法。
- 改変された遺伝子発現が、DNAまたはタンパク質マイクロアレイを用いて解析される、請求項1〜9のいずれか一項に記載の方法。
- 表現型解析が、代償として差異的に調節された遺伝子の発現を減少または除去させることによって行われる、請求項1〜10のいずれか一項に記載の方法。
- 代償として差異的に発現された遺伝子の発現が、コントロール生物に比べて増強された発現であり、減少または除去が、増強された発現を少なくとも部分的に阻害することによってなされる、請求項11に記載の方法。
- 差異的に発現された遺伝子のノックアウトが、差異的に調節された遺伝子のコード配列の少なくとも一部を、レポーター遺伝子のコード配列、または、レポーター遺伝子配列の検出されるのに十分な部分で置き換えることによって行われる、請求項7に記載の方法。
- 差異的に発現された遺伝子が、コントロール生物より強く発現されておらず、そして減
少または除去が、その発現を増強することによってなされる、請求項11に記載の方法。 - 減少または除去により、生物の成長阻害が起こる、請求項1〜14のいずれか一項に記載の方法。
- 表現型解析が、代償として差異的に調節された遺伝子の遺伝子産物を標識することによって行われる、請求項1〜10のいずれか一項に記載の方法。
- 請求項1〜16のいずれか一項に記載の方法によって得られる、遺伝学的に変化した表現型を有する生物。
- a)少なくとも1つの内因性または外来遺伝子の遺伝学的に改変された発現(それにより、生物にとって内因性の少なくとも1種のその他の遺伝子の代償としての差異的な発現が生じる)、および、
b)遺伝子の代償としての差異的な発現を減少/除去させることによって生じる表現型、または、代償として差異的に調節された遺伝子産物を標識することによって生じる表現型、
を有する、遺伝学的に改変された生物。 - 異種タンパク質またはタンパク質フラグメントの機能に対して作用する物質をスクリーニングするための、請求項17または18に記載の遺伝学的に改変された生物の使用。
- 請求項17または18に記載の生物の使用を含む、異種発現されたタンパク質またはタンパク質フラグメントの機能に対して作用する物質を同定する方法。
- 請求項17または18に記載の少なくとも1つの表現型を有する生物を用いる薬物スクリーニングのための分析であって:
b)該生物の表現型を決定する工程、
c)試験される物質と該生物とを接触させる工程、
d)該表現型の起こり得る改変を観察する工程、
を含む、上記分析。 - 請求項20に記載の方法、または、請求項21に記載の分析によって、表現型を少なくとも減少させる物質と同定された物質。
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JP5444553B2 (ja) | 2007-04-27 | 2014-03-19 | フェネックス インコーポレイテッド | 微生物宿主を迅速にスクリーニングして、異種タンパク質発現の収率および/または質が改善されている特定の株を同定する方法 |
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Non-Patent Citations (4)
Title |
---|
JPN5005013144, CAUMONT ANNE B, CRRENT GENETICS, 1996, V29 N6, P503−510 * |
JPN6009057213, Methods, 14[1](1998) p.35−42 * |
JPN6009057214, Trends Biotechnol., 15[12](1997) p.487−494 * |
JPN6009057215, Genome Research, 11[11](2001) p.1899−1912 * |
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