Photoactivatable Tet expression control system

Patent metadata

Field	Value
Publication number	US-12338443-B2
Application number	US-201917271037-A
Country	US
Kind code	B2
Filing date	Aug 30, 2019
Priority date	Aug 31, 2018
Publication date	Jun 24, 2025
Grant date	Jun 24, 2025

Abstract

The present invention provides a photoactivatable Tet-OFF/ON system that can precisely control temporal and spatial gene expression. The present invention is a PA-Tet-OFF/ON system that includes a target gene expression cassette including a TRE having a TetO sequence, a promoter which is controlled by the TRE, and a target gene whose expression is controlled by the promoter; a first fusion protein expression cassette containing a gene which encodes a first fusion protein containing TetR or rTetR and a first protein; and a second fusion protein expression cassette containing a gene which encodes a second fusion protein containing p65AD and a second protein, in which the first protein and the second protein bind to each other to form a heterodimer only in a state of being irradiated with light at a specific wavelength.

First claim

What is claimed is:

1. A photoactivatable tetracycline gene expression control system, comprising:
a target gene expression cassette including a tetracycline response element having a TetO sequence, a promoter which is positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a target gene which is positioned downstream of the promoter and of which expression is controlled by the promoter;
a first fusion protein expression cassette including a gene which encodes a first fusion protein containing a Tet repressor protein or a reverse Tet repressor protein and a first protein; and
a second fusion protein expression cassette including a gene which encodes a second fusion protein containing a transactivation domain of a transactivation element p65 and a second protein,
wherein the first protein and the second protein bind to each other and form a heterodimer only in a state of being irradiated with light at a specific wavelength, and
wherein the first protein is CIB1 or a variant thereof and the second protein is Cry2 or a variant thereof, or
wherein the first protein is Cry2 or a variant thereof and the second protein is CIB1 or a variant thereof, and
wherein the Tet repressor protein or the reverse Tet repressor protein has a threonine residue as an amino acid residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.

2. The photoactivatable tetracycline gene expression control system according to claim 1 ,
wherein in the first fusion protein, the Tet repressor protein or the reverse Tet repressor protein is linked to the first protein through a peptide linker consisting of an amino acid sequence represented by SPKKK.

3. The photoactivatable tetracycline gene expression control system according to claim 1 ,
wherein the first protein is CIB1 or a variant thereof, and the second protein is Cry2 or a variant thereof.

4. The photoactivatable tetracycline gene expression control system according to claim 3 ,
wherein Cry2 or a variant thereof contained in the second fusion protein is a C-terminal deletion variant having an N-terminal photolyase homology region or a variant obtained by substituting an amino acid residue in the C-terminal deletion variant with phenylalanine, and the amino acid residue corresponds to the 348th leucine of wild-type Cry2 of Arabidopsis thaliana.

5. The photoactivatable tetracycline gene expression control system according to claim 1 ,
wherein in the first fusion protein, CIB1 or a variant thereof is linked to a C-terminal side of the Tet repressor protein or the reverse Tet repressor protein.

6. The photoactivatable tetracycline gene expression control system according to claim 1 ,
wherein the CIB1 or a variant thereof contained in the first fusion protein is a C-terminal deletion variant of CIB1 that consists of a partial protein corresponding to a region consisting of the 1st to 170th amino acids of wild-type CIB1 of Arabidopsis thaliana or a variant that is obtained by deleting a nuclear localization signal from the C-terminal deletion variant of CIB1.

7. The photoactivatable tetracycline gene expression control system according to claim 6 ,
wherein CIB1 or a variant thereof contained in the first fusion protein is a variant obtained by deleting a nuclear localization signal from a C-terminal deletion variant of CIB1 consisting of a partial protein corresponding to a region consisting of the 1st to 170th amino acids of wild-type CIB1 of Arabidopsis thaliana , and
the second fusion protein contains a nuclear localization signal on the N-terminal or the C-terminal.

8. The photoactivatable tetracycline gene expression control system according to claim 1 , further comprising, in addition to the target gene expression cassette:
an expression cassette for a protein in which the first fusion protein and the second fusion protein are linked to each other through a T2A self-cleaving peptide; or
an expression cassette for bicistronically expressing the first fusion protein and the second fusion protein.

9. The photoactivatable tetracycline gene expression control system according to claim 1 ,
wherein the target gene is a gene that encodes a protein modified with ubiquitin.

10. A method for controlling target gene expression, comprising:
controlling expression of the target gene in a cell comprising the photoactivatable tetracycline gene expression control system according to claim 1 by adjusting conditions so that the cell is irradiated or not irradiated with blue light or near-infrared light and treated or not treated with a tetracycline-based compound.

11. A kit for a photoactivatable tetracycline gene expression control system, comprising:
a target gene expression vector including a tetracycline response element having a TetO sequence, a promoter which is positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a multicloning site which is positioned downstream of the promoter and into which a target gene will be inserted, and
an expression vector including an expression cassette for a protein in which the first fusion protein and the second fusion protein are linked to each other through a T2A self-cleaving peptide, or an expression cassette for bicistronically expressing the first fusion protein and the second fusion protein,
wherein the first fusion protein in which a Tet repressor protein or a reverse Tet repressor protein is linked to CIB1 or a variant thereof, and
wherein the second fusion protein in which a transactivation domain of a transactivation element p65 is linked to Cry2 or a variant thereof, and
wherein the Tet repressor protein or the reverse Tet repressor protein has a threonine residue as an amino acid residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.

12. A kit for a photoactivatable tetracycline gene expression control system, comprising:
a target gene expression vector including a tetracycline response element having a TetO sequence, a promoter which is positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a multicloning site which is positioned downstream of the promoter and into which a target gene will be inserted;
a first expression vector including a first fusion protein expression cassette containing a gene that encodes a first fusion protein in which a Tet repressor protein or a reverse Tet repressor protein is linked to CIB1 or a variant thereof, and
a second expression vector including a second fusion protein expression cassette containing a gene that encodes a second fusion protein in which a transactivation domain of a transactivation element p65 is linked to Cry2 or a variant thereof,
wherein the Tet repressor protein or the reverse Tet repressor protein has a threonine residue as an amino acid residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.

Assignees

• Japan Science & Tech Agency

Inventors

• Imayoshi Itaru
• Yamada Mayumi
• Suzuki Yusuke
• Nagasaki Shinji

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C07K14/4702

  Regulators; Modulating activity · CPC title

• C12N15/635

  Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline · CPC title

• C12N15/62 Primary

  DNA sequences coding for fusion proteins · CPC title

• C12N15/66 Primary

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title