Differentiation proteins

• US 20040209277A1
• Filed: 09/24/2003
• Published: 10/21/2004
• Est. Priority Date: 12/07/2001
• Status: Active Grant

• Current Assignee: Toolgen Incorporated
• Original Assignee: Toolgen Incorporated
• Inventors: Kim, Jin-Soo, Lee, Dong-Ki, Lee, Yangsoon
• Granted Patent: US 7,514,257 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/04 Plant cells or tissues culture media C12N5/0025
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02

Abstract

The disclosure describes artificial chimeric transcription factors that include at least one zinc finger domains and that can regulate cellular differentiation, for example, a neuronal cell phenotype or an osteoblast phenotype.

Claims

1. An isolated transcription factor that comprises at least one zinc finger domain, wherein presence of the transcription factor in a cell can alter the differentiation state of the cell.

2. The transcription factor of claim 1 wherein the cell is a vertebrate cell, and presence of the transcription factor in the cell can induce a neuronal phenotype in the cell.

3. The transcription factor of claim 2 wherein the cell is a vertebrate cell, and presence of the transcription factor in the cell can induce neurite extension in the cell.

4. The transcription factor of claim 2 wherein the DNA contacting residues of the zinc finger domain at positions −

1, [[1,]] 2, 3, and 6 of the domain correspond to a motif selected from the group consisting of;

QSNR, QSNK, and CSNR.

5. The transcription factor of claim 2 wherein the transcription factor further comprises a second and third zinc finger domain, and the DNA contacting residues of the first, second, and third domains at positions −

1, [[1,]] 2, 3, and 6 of each domain respectively correspond to the motifs;

QSNR, QSNK, and CSNR.

6. The transcription factor of claim 5 wherein the transcription factor comprises the amino acid sequence of the zinc finger array in SEQ ID NO:

2.

7. The transcription factor of claim 1 wherein the cell is a vertebrate cell, and presence of the transcription factor in the cell can induce expression of an osteoblast marker in the cell.

8. The transcription factor of claim 7 wherein the osteoblast marker is alkaline phosphatase.

9. The transcription factor of claim 7 wherein the cell is a myoblast cell line.

10. The transcription factor of claim 7 wherein the DNA contacting residues of the zinc finger domain at positions −

1, [[1,]] 2, 3, and 6 of the domain correspond to a motif selected from the group consisting of;

RDKR, QTHR, VSTR, and RDKR.

11. The transcription factor of claim 7 wherein the transcription factor further comprises a second and third zinc finger domain, and the DNA contacting residues of the first, second, and third domains at positions −

1, [[1,]] 2, 3, and 6 of each domain respectively correspond to (i) the motifs;

RDKR, QTHR, and VSTR or (ii) the motifs;

QTHR, VSTR, and RDKR.

12. The transcription factor of claim 11 wherein the transcription factor comprises the amino acid sequence of the zinc finger array in SEQ ID NO:

4.

13. A nucleic acid that encodes the transcription factor of claim 1.

14. A cell that contains the transcription factor of claim 1.

15. A method of altering the differentiation state of a cell, the method comprising:

expressing a nucleic acid that comprises a sequence encoding the protein of claim 1 in the cell; and

maintaining the cell under conditions that allow the protein to alter the differentiated state of the cell.

16. A method of altering the differentiation state of a vertebrate cell, the method comprising:

contacting the protein of claim 1 to the cell; and

maintaining the cell under conditions that allow the protein to alter the differentiated state of the cell.

17. A method of altering the differentiation state of a cell in a subject, the method comprising:

administering the protein of claim 1 or a nucleic acid that comprises a sequence encoding the protein to a subject in an amount effective to alter the differentiation state of at least one cell in the subject.

18. A method of inducing neurite formation in a vertebrate cell, the method comprising:

providing a vertebrate cell that contains a nucleic acid that comprises a sequence encoding an artificial transcription factor comprising three zinc finger domains, wherein expression of the artificial transcription factor induces a neuronal phenotype in the vertebrate cell; and

maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and neurite formation is induced.

19. The method of claim 18 wherein the DNA contacting residues of the first, second, and third domains at positions −

1, [[1,]] 2, 3, and 6 of each domain respectively correspond to the motifs;

QSNR, QSNK, and CSNR.

21. The method of claim 18 wherein the DNA contacting residues of the first, second, and third domains at positions −

1, [[1,]] 2, L and 6 of each domain respectively correspond to (i) the motifs;

RDKR, QTHR, and VSTR or (ii) the motifs;

QTHR, VSTR, and RDKR.

20. A method of inducing oosteogenesis in a vertebrate cell, the method comprising:

providing a vertebrate cell that contains a nucleic acid that comprises a sequence encoding an artificial transcription factor comprising three zinc finger domains, wherein expression of the artificial transcription factor induces oosteogenesis in the cell; and

maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and oosteogenesis is induced.