Tetracycline-regulated transcriptional activator fusion proteins

• US 20020086426A1
• Filed: 02/05/2001
• Published: 07/04/2002
• Est. Priority Date: 06/14/1993
• Status: Active Grant

• Current Assignee: Tet Systems Holding GmbH & Company Kg
• Original Assignee: BASF AG (BASF SE)
• Inventors: Voss, Jeffrey W., Salfeld, Jochen G., Gossen, Manfred, Bujard, Hermann
• Granted Patent: US 6,914,124 B2
• Time in Patent Office: Days
• US Class Current: 435/440
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/006 tet repressible

Abstract

A method for regulating expression of a tet operator-linked gene in a cell of a subject is disclosed. In one embodiment, the method involves introducing into the cell a nucleic acid molecule encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and modulating the concentration of a tetracycline, or analogue thereof, in the subject. Alternatively, in another embodiment, the method involves obtaining the cell from the subject, introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence, introducing into the cell a second nucleic acid molecule encoding a tTA, to form a modified cell, administering the modified cell to the subject, and modulating the concentration of a tetracycline, or analogue thereof, in the subject. The first and second nucleic acid molecule can be within a single molecule (e.g., in the same vector) or on separate molecules.

Claims

1. A method for regulating expression of a tet operator-linked gene in a cell of a subject, comprising:

introducing into the cell a nucleic acid molecule encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject.

2. The method of claim 1, wherein the Tet repressor of the tTA is a Tn10-derived Tet repressor.

3. The method of claim 1, wherein the polypeptide of the tTA which directly or indirectly activates transcription in eucaryotic cells is from herpes simplex virus virion protein 16.

4. The method of claim 1, wherein the nucleic acid molecule encoding the tTA is integrated randomly in a chromosome of the cell.

5. The method of claim 1, wherein the nucleic acid molecule encoding the tTA is integrated at a predetermined location within a chromosome of the cell.

6. The method of claim 1, wherein the nucleic acid molecule encoding the tTA is introduced into the cell ex vivo, the method further comprising administering the cell to the subject.

7. The method of claim 1, wherein the tet operator-linked gene is an endogenous gene of the cell which has been operatively linked to the at least one tet operator sequence.

8. The method of claim 1, wherein the tet operator-linked gene is an exogenous gene which has been introduced into the cells.

9. The method of claim 1, wherein the tetracycline analogue is anhydrotetracycline, doxycycline or cyanotetracycline.

10. A method for regulating expression of a gene in a cell of a subject, comprising:

obtaining the cell from the subject;

introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence;

introducing into the cell a second nucleic acid molecule encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells, to form a modified cell;

administering the modified cell to the subject; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject.

11. The method of claim 10, wherein the Tet repressor of the tTA is a Tn10-derived Tet repressor.

12. The method of claim 10, wherein the polypeptide of the tTA which directly or indirectly activates transcription in eucaryotic cells is from herpes simplex virus virion protein 16.

13. The method of claim 10, wherein the nucleic acid molecule encoding the tTA is integrated randomly in a chromosome of the cell.

14. The method of claim 10, wherein the nucleic acid molecule encoding the tTA is integrated by homologous recombination at a predetermined location within a chromosome of the cell.

15. The method of claim 10, wherein the first nucleic acid molecule operatively links an endogenous gene of the cell to at least one tet operator sequence.

16. The method of claim 10, wherein the first nucleic acid molecule comprises a gene operatively linked to at least one tet operator sequence.

17. The method of claim 10, wherein the tetracycline analogue is anhydrotetracycline, doxycycline or cyanotetracycline.