Method of concentrating and separating dopaminergic neurons

• US 7,270,998 B2
• Filed: 12/07/2000
• Issued: 09/18/2007
• Est. Priority Date: 06/01/2000
• Status: Expired due to Term

• Current Assignee: Japan Science And Technology Corpration
• Original Assignee: Japan Science And Technology Corpration
• Inventors: Okano, Hideyuki, Kobayashi, Kazuto, Sawamoto, Kazunobu, Matsushita, Natsuki
• Primary Examiner(s): Woitach; Joseph
• Assistant Examiner(s): Kelly; Robert M.
• Application Number:
  US10/048,536
• Publication Number:
  US 20020155423A1
• Time in Patent Office: 2,476 Days
• Field of Search: 536/23.1, 536/24.1, 424/93.1, 435/325, 435/455
• US Class Current: 435/325
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

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.

Claims

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, and

isolating 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.

2. The method as claimed in claim 1, wherein the fluorescent protein is a green fluorescent protein.

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.

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.

5. The method as claimed in claim 1, wherein the fluorescence-emitting cells are enriched and isolated by the use of a cell sorter.

6. A cell culture comprising cells that have been enriched and isolated by the method of claim 1.

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, and

measuring 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.

8. The method as claimed in claim 7, wherein the fluorescent protein is a green fluorescent protein.

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.

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.

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, and

determining 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.

12. The method as claimed in claim 11, wherein the fluorescent protein is a green fluorescent protein.

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.

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.

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, and

isolating 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.

16. The method as claimed in claim 15, wherein the fluorescent protein is a green fluorescent protein.

17. The method as claimed in claim 15, wherein the fluorescence-emitting cells are enriched and isolated by the use of a cell sorter.

18. A cell culture comprising the cells that have been enriched and isolated by the method of claim 15.

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, and

measuring 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.

20. The method as claimed in claim 19, wherein the fluorescent protein is a green fluorescent protein.