US 5,830,462 A · Regulated transcription of targeted genes and other biological events

Abstract

Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins. In principle, any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable, synthetic ligands. To illustrate the practice of this invention, we have induced: (1) the intracellular aggregation of the cytoplasmic tail of the .zeta. chain of the T cell receptor (TCR)-CD3 complex thereby leading to signaling and transcription of a reporter gene, (2) the homodimerization of the cytoplasmic tail of the Fas receptor thereby leading to cell-specific apoptosis (programmed cell death) and (3) the heterodimerization of a DNA-binding domain (Gal4) and a transcription-activation domain (VP16) thereby leading to direct transcription of a reporter gene.

Claims127

Dependent claims

A method for inducing a biological process in a cell which comprises the steps of:

(A) providing a cell comprising at least one genetic construct encoding a chimeric protein comprising (a) at least one ligand-binding domain which binds to a selected ligand, and (b) a heterologous protein domain which induces the biological process, wherein the selected ligand binds to and oligomerizes two or more chimeric protein molecules to form a ligand cross-linked complex and further has one or more of the following characteristics:
(i) the ligand is not a protein;
(ii) the ligand has a molecular weight less than 5 kD;
and
(iii) the ligand is membrane permeable,
and wherein mutual association of two or more of the heterologous protein domains in a ligand cross-linked complex induces the biological process;
and
(B) exposing the cell to the ligand in an amount effective to form the ligand cross-linked complex and thereby induce the biological process.

5 more elements

A method for providing a mammal responsive to a selected ligand which can regulate the transcription of a target gene in a cell within the mammal, which comprises introducing a cell into a host organism, wherein the cell comprises

(1) at least one genetic construct encoding a chimeric protein comprising (a) at least one ligand-binding domain which binds to a selected ligand, and (b) a heterologous protein domain which regulates transcription of the target gene, wherein the selected ligand binds to and oligomerizes two or more chimeric protein molecules to form a ligand cross-linked complex and further has one or more of the following characteristics:
(i) the ligand is not a protein;
(ii) the ligand has a molecular weight less than 5 kD;
and
(iii) the ligand is membrane permeable, and
(2) the target gene under the control of a transcription control element responsive to the ligand cross-linked complex.

3 more elements

A method for providing a mammal responsive to a ligand which can regulate the transcription of a target gene in a cell within the mammal, which comprises introducing at least one vector into a host mammal under conditions permitting transfection of one or more cells of the host mammal, wherein the vectors comprises

(1) at least one genetic construct encoding a chimeric protein comprising (a) at least one ligand-binding domain which binds to a selected ligand, and (b) a heterologous protein domain for regulating transcription of a target gene, wherein the selected ligand binds to and oligomerizes two or more chimeric protein molecules to form a ligand cross-linked complex and further has one or more of the following characteristics:
(i) the ligand is not a protein;
(ii) the ligand has a molecular weight less than 5 kD;
and
(iii) the ligand is membrane permeable, and
(2) a target gene under the control of a transcription control element responsive to the ligand cross-linked complex.

3 more elements

Dependencies are inferred from the claim text.

Regulated transcription of targeted genes and other biological events

Life of this patent

Regulated transcription of targeted genes and other biological events

Life of this patent

Abstract

Claims127