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Probe for visualizing cell-cycle UPDATE 実績あり

外国特許コード F190009901
整理番号 07219-US
掲載日 2019年8月26日
出願国 アメリカ合衆国
出願番号 45015508
公報番号 20100100977
公報番号 8182987
出願日 平成20年2月6日(2008.2.6)
公報発行日 平成22年4月22日(2010.4.22)
公報発行日 平成24年5月22日(2012.5.22)
国際出願番号 JP2008051973
国際公開番号 WO2008114544
国際出願日 平成20年2月6日(2008.2.6)
国際公開日 平成20年9月25日(2008.9.25)
優先権データ
  • 特願2007-068240 (2007.3.16) JP
  • 2008JP51973 (2008.2.6) WO
発明の名称 (英語) Probe for visualizing cell-cycle UPDATE 実績あり
発明の概要(英語) An object of an embodiment of the present invention is to provide a method with which it is possible to easily distinguish a proliferation phase of a cell cycle from a resting phase thereof in real time. The object of the embodiment of the present invention is attained by providing a method for performing phase identification of the cell cycle, the method including: visualizing one or more gene-expression products as markers whose amounts in a cell change in a cell-cycle dependent manner; and detecting the products so as to distinguish the proliferation phase of the cell cycle from the resting phase thereof.
従来技術、競合技術の概要(英語) BACKGROUND ART
A cell cycle is a process in which a cell produced by a cell division undergoes another cell division to produce a new cell. Of the cell cycle, a phase during which mitosis takes place is called an M phase. Generally, the M phase completes in approximately one hour. An interval between one M phase and another M phase is called an interphase during which cell growth as well as biosynthesis and/or metabolism of a substance occur. The interphase can be further divided into a G1 phase, an S phase, and a G2 phase. In the S phase, DNA replication takes place. The G1 phase is a phase between the M phase and the S phase, and the G2 phase is a phase between the S phase and the M phase.
As a method for analyzing a specific phase of the cell cycle (G1 phase, S phase, G2 phase, or M phase), a method using a BrdU label is known. In specific, the method includes: causing a BrdU to be taken into a cell for a given period; and subsequently, carrying out immunohistochemistry by using an anti-BrdU antibody. However, with the method, it is impossible to carry out observation in real-time. There is also known a method using cell synchronization and a biochemical model. With the method, however, it is impossible to carry out real-time observation.
As a method for visualizing a specific phase of the cell cycle, there is a method using the G2M cell cycle phase marker (G″ MCCPM) (Amersham Bioscience K.K). Because the method uses promoter activity of cyclin, there is a problem in that transformation by gene introduction is remarkably influenced depending on how a transgene is integrated into a chromosome. Further, because the G1 phase is not visualized, (i) it is difficult to track a cell cycle, and (ii) a contrast is unclear.
特許請求の範囲(英語) [claim1]
1. A method for performing phase identification of a cell cycle, the method comprising the steps of:
visualizing, by respectively using markers, at least two or more gene-expression products whose amounts in a cell change in a cell-cycle dependent manner; and
detecting the markers to distinguish a proliferation phase of the cycle from a resting phase of the cycle, wherein the at least two or more gene-expression products are co-expressed in a cell and comprise
(i) a first gene-expression product comprising a partial fragment of Cdt1; wherein the partial fragment of Cdt1 excludes a Geminin binding site, and increases in a G1 phase and decreases in an S/G2/M phase; and
(ii) a second gene-expression product comprising a partial fragment of Geminin; wherein the partial fragment of Geminin excludes a Cdt1 binding site, and decreases in the G1 phase and increases in the S/G2/M phase;
wherein the first gene-expression product and the second-gene expression product are labeled by the markers, and the markers are different from each other, to visualize the first gene-expression product and the second gene-expression product.

[claim2]
2. The method as set forth in claim 1, wherein the first gene-expression product is the partial fragment of Cdt1, which partial fragment of Cdt1 is composed of 30th through 120th amino acids of Cdt1.

[claim3]
3. The method as set forth in claim 1, wherein the second gene-expression product is the partial fragment of Geminin, which partial fragment of Geminin is composed of 1st through 110th amino acids of Geminin.

[claim4]
4. The method as set forth in claim 1, wherein the markers are a fluorescent protein or a luminescent protein.

[claim5]
5. The method as set forth in claim 1, wherein the markers are detected over time by carrying out a time-lapse imaging observation on a living cell or a living tissue.

[claim6]
6. The method as set forth in claim 1, wherein the first gene-expression product is labeled by a red fluorescent protein as the marker, and the second gene-expression product is labeled by a green fluorescent protein as the marker.

[claim7]
7. The method as set forth in claim 1, wherein the first gene-expression product is a product in which 1st through 30th amino acids of Cdt1 are further excluded.

[claim8]
8. The method as set forth in claim 1, further comprising
introducing into a cell (i) a first gene construct for encoding the first gene-expression product and the marker, and (ii) a second gene construct encoding the second gene-expression product and the marker, and
causing the first and second gene constructs respectively to express the first gene-expression product and marker labeling the first gene-expression product, and the second gene-expression product and marker labeling the second gene-expression product.

[claim9]
9. A method for screening a cell-cycle inhibitor or a drug for a cell cycle-related disease or a method for examining a compound, a drug, or a reagent on its effect and a functional mechanism, the method comprising the steps of:
incubating a cell in the presence of a candidate substance for the cell-cycle inhibitor, a candidate substance for the drug for the cell cycle-related disease, or a reagent for inhibiting specific gene expression; and
performing phase identification of a cell cycle in accordance with a method as set forth in claim 1, so as to select a candidate substance which has an influence on the cell cycle and/or cell death.
  • 発明者/出願人(英語)
  • Miyawaki Atsushi
  • Sawano Asako
  • Masai Hisao
  • RIKEN
  • TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE
国際特許分類(IPC)

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