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Probe for visualizing cell-cycle UPDATE_EN achieved

Foreign code F190009901
File No. 07219-US
Posted date Aug 26, 2019
Country United States of America
Application number 45015508
Gazette No. 20100100977
Gazette No. 8182987
Date of filing Feb 6, 2008
Gazette Date Apr 22, 2010
Gazette Date May 22, 2012
International application number JP2008051973
International publication number WO2008114544
Date of international filing Feb 6, 2008
Date of international publication Sep 25, 2008
Priority data
  • P2007-068240 (Mar 16, 2007) JP
  • 2008JP51973 (Feb 6, 2008) WO
Title Probe for visualizing cell-cycle UPDATE_EN achieved
Abstract 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.
Outline of related art and contending technology 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.
Scope of claims [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.
  • Inventor, and Inventor/Applicant
  • Miyawaki Atsushi
  • Sawano Asako
  • Masai Hisao
  • RIKEN
  • TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE
IPC(International Patent Classification)

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