Multiple inducible gene regulation system

a gene regulation and inducible technology, applied in the field of biotechnology or genetic engineering, can solve the problems of limited gene regulation at the moment, significant qualitative and quantitative limitations, and inability to regulate more than one gene at the same time in the same cell

Inactive Publication Date: 2008-09-25
RHEOGENE INC DE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, in applications that range from analyzing the human genome to proteomics to producing large-scale quantities of proteins, to gene therapies, there is no technology to regulate more than one gene at the same time in the same cell.
Yet, gene regulation at present is limited to one gene at a time, and this is a significant qualitative and quantitative limitation.
The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.
However, these systems have limitations due to their effect on expression of non-target genes.
These systems are also leaky.
Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e. tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression.
For similar reasons, utility of such systems in animals is limited.
Unfortunately, this system includes immunosuppressants that can have unwanted side effects which limits its use for various mammalian gene regulation system applications.
Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms.
Unfortunately, these USP-based systems are constitutive in animal cells and therefore, are not effective for regulating reporter gene expression.
Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands, non-applicability of these systems for use in both plants and animals (see U.S. Pat. No. 5,880,333), and limited use or inability to regulate expression of multiple genes.

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Examples

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example 1

[0277]This Example describes the construction of several gene expression cassettes for use in a multiple inducible gene expression system according to the invention. Applicants constructed several gene expression cassettes based on the spruce budworm Choristoneura fumiferana EcR (“CfEcR”), fruit fly Drosophila melanogaster EcR (“DmEcR”), green leafhopper Nephotetix cincticeps ecdysone receptor (“NeEcR”), mouse Mus musculus retinoid X receptor isoform α (“MmRXRα”), and locust Locusta migratoria invertebrate RXR homolog ultraspiracle protein (“LmUSP”). The prepared receptor constructs comprise a ligand binding domain of either an EcR or a vertebrate RXR; and a GAL4 DNA binding domain (DBD) or a VP16 transactivation domain (AD). The reporter constructs include a reporter gene, luciferase (Luc) or secreted alkaline phosphatase (SEAP), operably linked to a synthetic promoter construct that comprises a GALA response element to which the GAL4 DBD binds. Various combinations of these recept...

example 2

[0279]This Example describes the ability of a dual switch gene expression system of Applicants' invention to modulate expression of two reporter gene expression cassettes, wherein the two reporter gene expression cassettes are regulated independently by two different ligands. Specifically, one reporter gene expression cassette is inducibly regulated by a steroid ligand and the other reporter gene expression cassette is inducibly regulated by a non-steroid ligand. Briefly, Applicants prepared a dual switch inducible gene expression system as described above in Example 1.1. The resulting dual switch system was then tested in NIH3T3 mammalian cells as follows.

[0280]DNAs corresponding to the dual switch constructs outlined in Example 1.1 were transfected into mouse NIH3T3 cells (ATCC) as described in Example 1. At the end of the transfection incubation period, 250 μl of growth medium containing 20% FBS and either dimethylsulfoxide (DMSO; control) or a DMSO solution of 0.02, 0.1, 0.5, or...

example 3

[0282]This Example describes the ability of a dual switch gene expression system of Applicants' invention to modulate expression of two reporter gene expression cassettes, wherein the two reporter gene expression cassettes are regulated independently by two different ligands. In particular, one reporter gene expression cassette is inducibly regulated by a steroid ligand and the other reporter gene expression cassette is inducibly regulated by a non-steroid ligand. Briefly, Applicants prepared a dual switch inducible gene expression system as described above in Example 1.2. The resulting dual switch system was then tested in Chinese hamster ovary CHO cells as follows.

[0283]DNAs corresponding to the dual switch constructs outlined in Example 1.2 were transfected into hamster CHO cells (ATCC) as described in Example 1. CHO cells were transfected with 1) GAL4CfEcR-DEF / VP16MmRXRα-EF and pFRLuc, or 2) GAL4NcEcR-CDE / VP16MmRXRα-EF and pFRLuc. At the end of the transfection incubation period...

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Abstract

The present invention relates to the field of biotechnology or genetic engineering. More specifically, the present invention relates to a multiple inducible gene regulation system that functions within cells to simultaneously control the quantitative expression of multiple genes.

Description

RELATED APPLICATIONS[0001]This application claims priority to co-pending U.S. provisional application Ser. No. 60 / 237,446, filed Oct. 3, 2000, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]This invention relates to the field of biotechnology or genetic engineering. More specifically, the present invention relates to a multiple inducible gene regulation system that functions within cells, tissues, or organisms to simultaneously control the quantitative expression of two or more genes.BACKGROUND OF THE INVENTION[0003]Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.[0004]In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and oth...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C07H21/00C12N15/00C12N1/20A01H5/00C12N5/04C12N1/00C12N5/06C07K14/72C12N1/15C12N1/19C12N1/21C12N5/10C12N7/00C12N15/09C12N15/63C12N15/82
CPCA01K2217/05G01N33/6845C07K2319/00C12N15/635C12N15/8217C12N15/8238C12N2799/021C12N15/85C12N15/861C12N15/70C12N15/74C12N15/80C12N15/81C12N15/8241C07K14/721A61P1/00A61K48/00
Inventor DHADIALLA, TARLOCHAN SINGHCRESS, DEAN ERVINCARLSON, GLEN MARC RICHARDHORMANN, ROBERT EUGENEPALLI, SUBBA REDDYKUDLA, ARTHUR JOHNHERZIG, RONALD PHILLIPPHILIP, MOHAN
Owner RHEOGENE INC DE
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