Methods and Compositions for Regulating Gene Expression

Abstract

In certain embodiments, the disclosure relates to compositions and methods relating to a translation-based gene regulation system that functions in mammalian cells. In certain specific embodiments, the disclosure relates to methods of regulating gene expression via modulating translational termination.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of the filing date of U.S. Provisional Application 60 / 727,327, filed Oct. 17, 2005. The entire teachings of the referenced provisional application are expressly incorporated by reference herein.FUNDING[0002]This invention was made with government support under Grant Number NIH 5PO-HL54785 awarded by National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]The ability to exogenously control the expression of genes in mammalian cells has been a powerful tool of biomedical research. In particular, gene regulation technology has played a central role in efforts to understand the role of specific gene products in fundamental biological processes and in both normal development and disease states. It is likely that this form of genetic technology will continue to have impact in a variety of areas of basic research and may enable new therapeutic paradigm...

AI Technical Summary

Benefits of technology

[0017]In certain embodiments, the present invention provides a method for identifying an agent that suppresses a nonsense mutation. Such method comprises (a) providing a test host cell which contains a polynucleotide molecule comprising a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein; (b) providing a control host cell comprising a wild type nucleic acid encoding the target protein expressed under an appropriate promoter and the expression of the target protein is at wild type level; (c) contacting the test host cell and the control host cell with a candidate agent; and (d) assaying for the level of the target protein in the test host cell and in control host cell. If the candidate agent induces translation of the target protein in the test host cell to a significant level, the candidate agent is an agent that suppresses the nonsense mutation. Optionally, the target protein is fused to a reporter moiety and the expression of the reporter moiety is assayed. A preferred host cell is a mammalian cell. In certain cases, the number of codons between the nonsense mutation and the start codon is selected from: 0, 1, 2, 3, 4 and 5. In certain specific embodiments, the target protein is a secreted protein, and the nonsense mutation is introduced in a region of the nucleic acid which encodes a signal peptide.

[0018]In certain embodiments, the present invention provides a method for identifying an agent that suppresses a nonsense mutation in vivo. Such method comprises (a) providing a transgenic nonhuman animal which contains a polynucleotide molecule comprising a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein; (b) providing a control nonhuman animal which contains a wild type nucleic acid encoding the target protein expressed under an appropriate promoter and the expression of the target protein is at wild type level; (c) administering to the tra

Problems solved by technology

Despite their considerable utility, these systems possess some significant limitations due to their reliance on chimeric transcriptional transactivators and specialized promoter elements.

Such limitations include the requirement for co-introduction of genes encoding the relevant transcriptional transactivator along with the gene to be regulated, and the inability to provide for the “on-off” regulation of a gene in the context of its own endogenous transcriptional control elements.

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