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Probe for visualizing cell cycle UPDATE_EN achieved

Foreign code F190009891
File No. 07219-EP
Posted date Aug 26, 2019
Country EPO
Application number 08710869
Gazette No. 2138577
Gazette No. 2138577
Date of filing Feb 6, 2008
Gazette Date Dec 30, 2009
Gazette Date Mar 22, 2017
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 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 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.
Prior art document Xouri G et al. 2007. Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin. EMBO J. 26:1303-1314 describes that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. It also describes that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin.
Prior art document Ballabeni et al. 2004. Human Geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis. EMBO J. 23:3122-3132 describes that Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis and that consistently inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. In addition, it describes that inhibition of CDR1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells.
Yoshida K & Inoue I. 2004. Peptide binding to Geminin and inhibitory for DNA replication. BBRC. 317: 218-222 describes that delivery of a peptide sequence that binds the 31-111 amino acid residues of Germinin into the nucleus of HCT116 human colon cancer cells resulted in the suppression of BrdU incorporation.
Benjamin JM. 2004. Geminin Has Dimerization, dtl-binding, and Destruction Domains That Are Required for Biological Activity. 279(44): 45957-45968 described an investigation into the requirement for certain domains of Gerinin for biological activity.
Xouri G et al. 2004. Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell lines. 271:3368-3378 described the expression levels of Germinin in cancer cells.
Scope of claims [claim1]
1. A method for performing phase identification of a cell cycle, the method comprising the steps of:
visualizing, at least two or more gene-expression products whose amounts in a cell change in a cell-cycle dependent manner; and
detecting in vitro the at least two or more gene-expression products so as to distinguish a proliferation phase of the cell cycle from a resting phase of the cell cycle, wherein:
as the at least two or more gene-expression products, at least (i) a first gene-expression product whose amount increases in a G1 phase and decreases in an S/G2/M phase and (ii) a second gene-expression product whose amount decreases in the G1 phase and increases in the S/G2/M phase are used in one cell;
the first gene-expression product is the partial fragment of Cdt1, which partial fragment of Cdt1 is remaining of Cdt1 from which a Geminin binding site is excluded;
the second gene-expression product is the partial fragment of Geminin, which partial fragment of Geminin is remaining of Geminin from which a Cdt1 binding site is excluded, and
the first gene-expression product and the second gene-expression product being labeled by the markers different from each other, so as to enable the first gene-expression product and the second gene-expression product to be visualized and detected, 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, and 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.

[claim2]
2. The method as set forth in claim 1, wherein the marker is a fluorescent protein or a luminescent protein.

[claim3]
3. The method as set forth in any one of Claims 1 or 2, wherein the marker is detected over time by carrying out a time-lapse imaging observation on a living cell or a living tissue.

[claim4]
4. A transformed cell or a transgenic nonhuman animal, coexpressing in a cell, (i) a first gene construct including a gene encoding a marker, and a gene encoding a partial fragment of Cdt1 which is remaining of Cdt1 from which a Geminin binding site is excluded and (ii) a second gene construct including a gene encoding a marker, and a gene encoding a partial fragment of Geminin which is remaining of Geminin from which a Cdt1 binding site is excluded,
the markers being different from each other, wherein
the gene encoding a partial fragment of Cdt1which is remaining of Cdt1 from which a Geminin binding site is excluded is a gene encoding a partial fragment of Cdt1 composed of 30th through 120th amino acids of said Cdt1, and wherein
the gene encoding a partial fragment of Geminin which is remaining of Geminin from which a Cdt1 binding site is excluded is a gene encoding a partial fragment of Geminin composed of 1st through 110th amino acids of said Geminin.

[claim5]
5. The transformed cell or the transgenic nonhuman animal as set forth in Claim 4, wherein:
the gene encoding the marker is a gene encoding a fluorescent protein or a luminescent protein.

[claim6]
6. A method for in vitro screening a cell-cycle inhibitor comprising the steps of:
incubating a cell in the presence of a candidate substance for the cell-cycle inhibitor, and
performing phase identification of a cell cycle in accordance with a method as set forth in any one of Claims 1 through 3 , so as to select a candidate substance for inducing cell cycle arrest.

[claim7]
7. A method for performing phase identification of a cell cycle as set forth in claim 1, wherein:
the marker for the first gene-expression product is a red fluorescent protein, and the marker for the second gene-expression product is a green fluorescent protein.

[claim8]
8. A transformed cell or a transgenic nonhuman animal as set forth in Claim 4, wherein the one of the markers encoded by the first gene construct is a red fluorescent protein and the other one encoded by the second gene construct is a green fluorescent protein.

[claim9]
9. A probe for visualising cell cycle comprising:
a first gene construct including a gene encoding a marker, and a gene encoding a partial fragment of Cdt1 which is remaining of Cdt1 from which a Geminin binding site is excluded; and
a second gene construct including a gene encoding a marker, and a gene encoding a partial fragment of Geminin which is remaining of Geminin from which a Cdt1 binding site is excluded,
the markers being different from each other.

[claim10]
10. The probe for visualising cell cycle as set forth in Claim 9, wherein the marker encoded by the first gene construct is a red fluorescent protein and the marker encoded by the second gene construct is a green fluorescent protein.
  • Applicant
  • RIKEN
  • TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE
  • Inventor
  • MIYAWAKI, Atsushi
  • SAWANO, Asako
  • MASAI, Hisao
IPC(International Patent Classification)
Specified countries Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

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