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Method of concentrating and separating dopaminergic neurons 新技術説明会 実績あり

外国特許コード F110005003
整理番号 A011-09US
掲載日 2011年8月8日
出願国 アメリカ合衆国
出願番号 04853600
公報番号 20020155423
公報番号 7270998
出願日 平成12年12月7日(2000.12.7)
公報発行日 平成14年10月24日(2002.10.24)
公報発行日 平成19年9月18日(2007.9.18)
国際出願番号 JP2000008674
国際公開番号 WO2001092482
国際出願日 平成12年12月7日(2000.12.7)
国際公開日 平成13年12月6日(2001.12.6)
優先権データ
  • 特願2000-165150 (2000.6.1) JP
  • 2000WO-JP08674 (2000.12.7) WO
発明の名称 (英語) Method of concentrating and separating dopaminergic neurons 新技術説明会 実績あり
発明の概要(英語) (US7270998)
The invention of this application provides a method comprising introducing a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a gene that is expressed in dopaminergic neurons, into each of cells, and isolating fluorescence-emitting cells.
The invention also provides a method for visualizing and identifying dopaminergic neurons alive that exist with in cells, which comprises introducing the above-mentioned reporter nucleic acid molecule into each of cells, and measuring the fluorescence distribution within the cells.
The invention further provides a method for identifying a dopaminergic neurons-inducing factor, which comprises introducing the reporter nucleic acid molecule into cells that have the ability to differentiate into dopaminergic neurons, then incubating the cells with a candidate substance, and determining whether the candidate substance is a dopaminergic neurons-inducing factor by using the fluorescence of the cells as an indicator.
特許請求の範囲(英語) [claim1]
1. A method for enriching and/or isolating dopaminergic neurons from a non-human transgenic animal or its progeny, which comprises: introducing a recombinant vector comprising a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a tyrosine hydroxylase gene of the non-human animal into a fertilized egg of the non-human animal,developing the non-human fertilized egg whose genome comprises the reporter nucleic molecule into a non-human transgenic animal by transfer to a surrogate mother, andisolating cells that emit a fluorescent signal from a plurality of cells of the non-human transgenic animal or its progeny whose genome comprises the reporter nucleic acid molecule, wherein the fluorescence-emitting cells are dopaminergic neurons.
[claim2]
2. The method as claimed in claim 1, wherein the fluorescent protein is a green fluorescent protein.
[claim3]
3. The method as claimed in claim 1, wherein the plurality of cells are derived from brain of the non-human transgenic animal or its progeny.
[claim4]
4. The method as claimed in claim 1, wherein the plurality of cells are derived from marrow mesenchymal cells of the non-human transgenic animal or its progeny.
[claim5]
5. The method as claimed in claim 1, wherein the fluorescence-emitting cells are enriched and isolated by the use of a cell sorter.
[claim6]
6. A cell culture comprising cells that have been enriched and isolated by the method of claim 1.
[claim7]
7. A method for identifying live dopaminergic neurons from a non-human transgenic animal or its progeny, which comprises: introducing a recombinant vector comprising a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a tyrosine hydroxylase gene of the non-human animal into a fertilized egg of the non-human animal,developing the non-human fertilized egg whose genome comprises the reporter nucleic acid molecule into a non-human transgenic animal by transfer to a surrogate mother, andmeasuring the fluorescence distribution within a plurality of cells obtained from the non-human animal or its progeny whose genome comprises the reporter nucleic acid molecule, wherein the fluorescence is an indicator of live dopaminergic neurons.
[claim8]
8. The method as claimed in claim 7, wherein the fluorescent protein is a green fluorescent protein.
[claim9]
9. The method as claimed in claim 7, wherein the plurality of cells are derived from brain of the non-human transgenic animal or its progeny.
[claim10]
10. The method as claimed in claim 7, wherein the plurality of cells are derived from marrow mesenchymal cells of the non-human transgenic animal or its progeny.
[claim11]
11. A method for identifying a factor which induces cells to differentiate into dopaminergic neurons, which comprises: introducing a recombinant vector comprising a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a tyro sine hydroxylase gene of the non-human animal into a fertilized egg of the non-human animal,developing the non-human fertilized egg whose genome comprises the reporter nucleic acid molecule into a non-human transgenic animal by transfer to a surrogate mother,incubating cells obtained from the non-human transgenic animal or its progeny whose genome comprises the reporter nucleic acid molecule with a candidate substance, anddetermining whether the candidate substance is the dopaminergic neuron-inducing factor by identifying cells that emit a fluorescent signal, greater than background fluorescence to indicate the presence of the dopaminergic neuron-inducing factor.
[claim12]
12. The method as claimed in claim 11, wherein the fluorescent protein is a green fluorescent protein.
[claim13]
13. The method as claimed in claim 11, wherein the cells that have an ability to differentiate into dopaminergic neurons are neural stem cells of the non-human transgenic animal or its progeny.
[claim14]
14. The method as claimed in claim 11, wherein the cells that have an ability to differentiate into dopaminergic neurons are marrow mesenchymal cells of the non-human transgenic animal or its progeny.
[claim15]
15. A method for enriching and/or isolating dopaminergic neurons from a transgenic mouse or its progeny, which comprises: introducing a recombinant vector comprising a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a tyro sine hydroxylase gene of the mouse or the promoter/enhancer of a tyrosine hydroxylase gene obtained from a rat into mouse ES cells,developing the mouse ES cells whose genome comprises the reporter nucleic acid molecule into a transgenic mouse by introducing the ES cells into a blastocyst and transferring the blastocyst to a surrogate mother, andisolating cells that emit a fluorescent signal from a plurality of cells obtained from the transgenic mouse or its progeny whose genome comprises the reporter nucleic acid molecule, wherein the fluorescence-emitting cells are dopaminergic neurons.
[claim16]
16. The method as claimed in claim 15, wherein the fluorescent protein is a green fluorescent protein.
[claim17]
17. The method as claimed in claim 15, wherein the fluorescence-emitting cells are enriched and isolated by the use of a cell sorter.
[claim18]
18. A cell culture comprising the cells that have been enriched and isolated by the method of claim 15.
[claim19]
19. A method for identifying live dopaminergic neurons from a transgenic mouse or its progeny, which comprises: introducing a recombinant vector comprising a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a tyrosine hydroxylase gene of the mouse or the promoter/enhancer of a tyrosine hydroxylase gene obtained from a rat into mouse ES cells,developing the mouse ES cells whose genome comprises the reporter nucleic acid molecule into a transgenic mouse by introducing the ES cells into a blastocyst and transferring the blastocyst to a surrogate mother, andmeasuring the fluorescence distribution within a plurality of cells obtained from the transgenic mouse or its progeny whose genome comprises the reporter nucleic acid molecule, wherein the fluorescence is an indicator of live dopaminergic neurons.
[claim20]
20. The method as claimed in claim 19, wherein the fluorescent protein is a green fluorescent protein.
  • 発明者/出願人(英語)
  • OKANO HIDEYUKI
  • SAWAMOTO KAZUNOBU
  • KOBAYASHI KAZUTO
  • MATSUSHITA NATSUKI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 435/325
  • 435/4
  • 800/3
  • 800/8
参考情報 (研究プロジェクト等) CREST Understanding the Brain AREA
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