Photoactivatable Tet expression control system
Inventors
IMAYOSHI, Itaru • Yamada, Mayumi • Suzuki, Yusuke • NAGASAKI, Shinji
Assignees
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Publication Number
US-12338443-B2
Publication Date
2025-06-24
Expiration Date
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.
Core Innovation
The document describes a photoactivatable tetracycline gene expression control system in which a target gene expression cassette includes a tetracycline response element having a TetO sequence, with a promoter positioned downstream of the tetracycline response element and controlled by the tetracycline response element. A target gene is positioned downstream of the promoter, and expression of the target gene is controlled by the promoter.
The system includes a first fusion protein expression cassette encoding a first fusion protein that contains either a Tet repressor protein or a reverse Tet repressor protein and a first protein, and a second fusion protein expression cassette encoding a second fusion protein that contains a transactivation domain of a transactivation element p65 and a second protein. 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.
The document specifies that the first protein is CIB1 or a variant thereof and the second protein is Cry2 or a variant thereof, or alternatively that the first protein is Cry2 or a variant thereof and the second protein is CIB1 or a variant thereof. The Tet repressor protein or reverse Tet repressor protein includes a threonine residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.
Claims Coverage
The provided material includes three independent claims. Each independent claim centers on a TetO-regulated target expression cassette and light-dependent heterodimerization between two fusion proteins, with an engineered residue in the Tet repressor or reverse Tet repressor.
Photoactivatable tetracycline gene expression control system with light-dependent heterodimerization
A photoactivatable tetracycline gene expression control system comprising a target gene expression cassette including a tetracycline response element having a TetO sequence, a promoter positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a target gene positioned downstream of the promoter and whose expression is controlled by the promoter; a first fusion protein expression cassette encoding 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 encoding 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; 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.
Kit with TetO-regulated target vector and T2A/bicistronic fusion expression vectors for photoactivatable Tet control
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 positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a multicloning site positioned downstream of the promoter and into which a target gene will be inserted; an expression vector including an expression cassette for a protein in which the first fusion protein and the second fusion protein are linked 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 is a Tet repressor protein or a reverse Tet repressor protein linked to CIB1 or a variant thereof; and wherein the second fusion protein is a transactivation domain of a transactivation element p65 linked to Cry2 or a variant thereof; wherein the Tet repressor protein or the reverse Tet repressor protein has a threonine residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.
Kit with separate target vector and two fusion expression vectors for photoactivatable Tet control
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 positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a multicloning site 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 encoding 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 encoding 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 corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.
Across the independent claims, the coverage centers on TetO-regulated promoter control of a target gene, combined with light-dependent heterodimerization between fusion proteins comprising CIB1 or Cry2 variants, and an engineered Tet repressor or reverse Tet repressor threonine residue corresponding to the 194th isoleucine of wild-type Escherichia coli Tet repressor.
Stated Advantages
Documented Applications
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