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Artificial mammalian chromosome 実績あり

外国特許コード F110003229
整理番号 A022-23EP
掲載日 2011年6月22日
出願国 欧州特許庁(EPO)
出願番号 03794154
公報番号 1536007
公報番号 1536007
出願日 平成15年9月1日(2003.9.1)
公報発行日 平成17年6月1日(2005.6.1)
公報発行日 平成27年12月30日(2015.12.30)
国際出願番号 JP2003011134
国際公開番号 WO2004022741
国際出願日 平成15年9月1日(2003.9.1)
国際公開日 平成16年3月18日(2004.3.18)
優先権データ
  • 2003JP011134 (2003.9.1) WO
  • 特願2002-258114 (2002.9.3) JP
  • 特願2002-338865 (2002.11.22) JP
発明の名称 (英語) Artificial mammalian chromosome 実績あり
発明の概要(英語) It is intended to provide an artificial mammalian chromosome which is stably held in mammalian cells and allows efficient expression of a target gene carried thereby.
Namely, a first cyclic vector containing a mammalian centromere sequence and a selection marker gene and a second cyclic vector containing a functional sequence are transferred into mammalian host cells.
Then transformed cells are selected by using the above-described selection marker gene and cells holding an artificial mammalian chromosome are selected from among the transformed cells thus selected.
Thus, it is possible to construct an artificial mammalian chromosome which has a mammalian replication origin, the mammalian centromere sequence and the functional sequence, is in a cyclic form, can be replicated in mammalian cells, extrachromosomally held in the host cells and transferred to daughter cells in cell division. <IMAGE>
特許請求の範囲(英語) [claim1]
1. A production method of a mammalian artificial chromosome, comprising: a first step of introducing into a mammalian host cell a first vector being circular in form and comprising a mammalian centromere sequence and a second vector being circular in form and comprising an insertion sequence for specifically inserting a sequence of interest and comprising an insulator sequence; a second step of selecting transformed cells; and a third step of selecting a cell containing a mammalian artificial chromosome from the selected transformed cells, wherein the mammalian cell is not a human embryonic cell.
[claim2]
2. A production method of a mammalian artificial chromosome, comprising: a first step of introducing into a mammalian host cell a first vector consisting of a yeast artificial chromosome having a mammalian centromere sequence and a mammalian telomere sequence and a second vector consisting of a yeast artificial chromosome having an insertion sequence for specifically inserting a sequence of interest and comprising an insulator sequence; a second step of selecting transformed cells; and a third step of selecting a cell containing a mammalian artificial chromosome from the selected transformed cells, wherein the mammalian cell is not a human embryonic cell.
[claim3]
3. The production method according to claim 1 or 2, wherein the first vector has a selection marker gene and the selection of the transformed cells in the second step is carried out by using the selection marker gene.
[claim4]
4. The production method according to any of claims 1 to 3, wherein the mammalian centromere sequence comprises a region in which a plurality of the following sequences are arranged at regular intervals: 5'-NTTCGNNNNANNCGGGN-3': SEQ ID NO. 1, wherein N is selected from the group consisting of A, T, C and G.
[claim5]
5. The production method according to any of claims 1 to 4, wherein the mammalian centromere sequence comprises a sequence derived from a human chromosome alpha satellite region.
[claim6]
6. The production method according to claim 5, wherein the mammalian centromere sequence comprises a 11mer repeat unit derived from a human chromosome 21.
[claim7]
7. The production method according to any of claims 1 to 6, wherein the size of the mammalian centromere sequence is about 50 kb or less.
[claim8]
8. The production method according to any of claims 1 to 7, wherein the insertion sequence is a loxP site, a FRT site, or a sequence obtained by partial modification of a loxP site or a FRT site and has a function for inserting the sequence of interest.
[claim9]
9. The production method according to any of claims 1 to 8, wherein the quantity ratio of the first vector to the second vector, which are inserted in the first step, is in the range from about 10 : 1 molecular ratio to about 1 : 10 molecular ratio.
[claim10]
10. A mammalian artificial chromosome obtainable by the production method described in any of claims 1 to 9, which comprises (a) a mammalian replication origin, a mammalian centromere sequence, an insertion sequence for specifically inserting a sequence of interest and an insulator sequence, and
which is circular in form and is replicated in a mammalian cell, maintained extrachromosomally in a host cell, and transmitted to daughter cells during cell division; or (b) a mammalian replication origin, a mammalian centromere sequence, a mammalian telomere sequence, an insertion sequence for specifically inserting a sequence of interest and an insulator sequence, and
which is linear in form and is replicated in a mammalian cell, maintained extrachromosomally in a host cell, and transmitted to daughter cells during cell division.
[claim11]
11. The mammalian artificial chromosome according to claim 10, wherein the insertion sequence is a loxP site, a FRT site, or a sequence obtained by partial modification of a loxP site or a FRT site and has a function for inserting the sequence of interest.
[claim12]
12. The mammalian artificial chromosome according to claim 10 or 11, wherein the mammalian centromere sequence comprises a region in which a plurality of the following sequences are arranged at regular intervals: 5'-NTTCGNNNNANNCGGGN-3': SEQ ID NO. 1, wherein N is selected from the group consisting of A, T, C and G.
[claim13]
13. The mammalian artificial chromosome according to any of claims 10 to 12, wherein the mammalian centromere sequence comprises a sequence derived from a human chromosome alpha satellite region.
[claim14]
14. The mammalian artificial chromosome according to claim 13, wherein the mammalian centromere sequence comprises an 11mer repeat unit derived from a human chromosome 21.
[claim15]
15. The mammalian artificial chromosome according to any of claims 10 to 14, comprising a plurality of the sequences of interest or the insertion sequences.
[claim16]
16. A mammalian cell containing the mammalian artificial chromosome described in any of claims 10 to 15 outside the autonomous chromosome, wherein the mammalian cell is not a human embryonic cell.
[claim17]
17. The mammalian cell of claim 16, which is a human cell.
[claim18]
18. A non-human embryonic stem cell containing the mammalian artificial chromosome described in any of claims 10 to 15 outside the autonomous chromosome.
[claim19]
19. An in vitro method of stably maintaining the sequence of interest or the insertion sequence for a long term in a mammalian cell, the method comprising: introducing the mammalian artificial chromosome of any one of claims 10 to 15 into mammalian cells as target, wherein the mammalian cell is not a human embryonic cell.
[claim20]
20. A production method of a mammalian cell containing a mammalian artificial chromosome, the method comprising the steps of the method of any of claims 1 to 9, and
a fourth step of isolating the mammalian artificial chromosome of any one of claims 10 to 15 from the selected cells; and
a fifth step of introducing the isolated mammalian artificial chromosome into a mammalian cell as a target cell, wherein the mammalian cell is not a human embryonic cell.
[claim21]
21. A production method of a micro-cell containing a mammalian artificial chromosome, the method comprising the steps of the method of any of claims 1 to 9, and
a fourth step of fusing the selected cell with a mammalian cell having an ability of forming micro-cells;
a fifth step of selecting a hybrid cell capable of forming micro-cells and containing the mammalian artificial chromosome of any one of claims 10 to 15; and
a sixth step of forming micro-cells from the selected hybrid cell, wherein said micro-cells contain said mammalian artificial chromosome.
[claim22]
22. A production method of a mammalian cell containing a mammalian artificial chromosome, comprising: isolating the mammalian artificial chromosome from a host cell containing the mammalian artificial chromosome of any of claims 10 to 15; and introducing the isolated mammalian artificial chromosome into a mammalian cell as a target cell, wherein the mammalian cell is not a human embryonic cell.
[claim23]
23. A production method of a micro-cell containing a mammalian artificial chromosome, the method comprising: fusing a host cell containing the mammalian artificial chromosome of any of claims 10 to 15 and a mammalian cell having an ability of forming micro-cells; selecting a hybrid cell having an ability of forming micro-cells and containing the mammalian artificial chromosome; and forming micro-cells from the selected hybrid cells, wherein said micro-cells contain said mammalian artificial chromosome.
[claim24]
24. A production method of a mammalian cell containing a mammalian artificial chromosome, the method comprising: fusing the micro-cell obtainable by the production method of claim 21 or 23 with a mammalian cell as a target, wherein the mammalian cell is not a human embryonic cell.
[claim25]
25. The production method of a mammalian cell according to any of claims 19, 20, 22 and 24, wherein the mammalian cell as a target cell is a non-human embryonic stem cell, non-human embryonic germ cell, tissue stem cell, or a non-human fertilized egg.
[claim26]
26. The production method of a mammalian cell according to any of claims 19, 20, 22 and 24, wherein the mammalian cell as a target cell is formed by inducing a non-human embryonic stem cell, non-human embryonic germ cell, or tissue stem cell so as to be differentiated to a cell of specific tissue.
[claim27]
27. Use of a vector for producing a mammalian artificial chromosome in a method according to any of claims 1 to 9, said vector comprising: a sequence of a loxP site or FRT site, or a sequence obtainable by partial modification of a loxP site or FRT site, the sequence having a function for inserting the sequence of interest, and an insulator sequence.
[claim28]
28. A transformed mouse comprising the mammalian artificial chromosome of claim 10.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • OKAZAKI TSUNEKO
  • IKENO MASASHI
  • ITOU TOSHIHIDE
  • SUZUKI NOBUTAKA
国際特許分類(IPC)
欧州特許分類/主・副
  • C12N015/85
  • K01K217/05
  • K61K048/00
  • M12N800/20B
  • M12N800/20C
  • M12N800/20D
  • M12N820/00
  • M12N820/85
指定国 Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR
参考情報 (研究プロジェクト等) CREST Genetic Programming AREA
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