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Methods for modulating expression of exogenous genes in mammalian systems, and products related thereto

a technology of exogenous genes and products, applied in the field of recombinant dna technology, can solve the problems of slow clearance of antibiotics from bone, tetracycline pharmacokinetics may hinder its use during development, etc., and achieves convenient storage, rapid clearance, and precise and potent induction

Inactive Publication Date: 2006-01-19
EVANS RONALD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] Unlike prior art tetracycline based strategies, transferring ecdysone responsiveness to mammalian cells takes advantage of a naturally evolved steroid inducible system. Advantages of ecdysteroid use include the lipophilic nature of the compounds (which provides efficient penetrance thereof into all tissues, including the brain), short half-lives (which allow for precise and potent inductions), and favorable pharmacokinetics that prevent storage and expedite clearance.

Problems solved by technology

However, disadvantages of this system include the continuous treatment of tetracycline to repress expression and the slow clearance of antibiotic from bone which interferes with: quick and precise induction.
While this system has been improved by the recent identification of a mutant tetracycline repressor which acts conversely as an inducible activator, the pharmacokinetics of tetracycline may hinder its use during development when a precise and efficient “on-off” switch is essential (Gossen et al.

Method used

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  • Methods for modulating expression of exogenous genes in mammalian systems, and products related thereto
  • Methods for modulating expression of exogenous genes in mammalian systems, and products related thereto
  • Methods for modulating expression of exogenous genes in mammalian systems, and products related thereto

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Modified Ecdysone Receptors

Plasmid Preparation:

[0176] The plasmids CMX-EcR, CMX-USP, CMX-FXR, CMX-hRXRa, EcREx5-ΔMTV-Luc, CMX-GEcR, MMTV-luc, and CMX-GR have been previously described (Yao, et al., Nature 366:476-479 (1993) and Forman, et al. Cell 81:687-693 (1995)).

[0177] The plasmid CMX-VpEcR was constructed by ligation of an EcoRI fragment of psk-EcR and CMX-Vp16.

[0178] The plasmid CMX-VgEcR was generated by site-directed mutagenesis of CMX-VpEcR using the Transformer Mutagenesis Kit (Clontech) and the mutagenic oligonucleotide (SEQ ID NO:14): [0179] 5′-TACAACGCCCTCACCTGTGGATCCTGCAAGGTGTTTCTTTCGACGCAGC-3′.

Mutagenesis of VpEcR to VgEcR altered the P-box region of the DNA binding domain of ecdysone receptor to resemble that of GR (Umesono and Evans, Cell 57:1139-1146 (1989)). The following amino acids in the DNA-binding domain of the ecdysone receptor were altered: E282G, G283S, and G286V (E=glutamate, G=glycine, S=serine, V=valine).

[0180] The reporter constr...

example 2

Construction of a Novel Ecdysone Response Element

[0192] Although no mammalian transcription factors have been shown to have a natural enhancer element like the ecdysone response element, which is composed of two inverted half-sites of the sequence AGGTCA spaced by one nucleotide, it is difficult to preclude such a possibility. The recently cloned farnesoid X receptor (FXR) can very weakly activate certain synthetic promoters containing an ecdysone response element in response to extremely high concentrations of farnesoids (Forman et al., Cell 81:687-693 (1995)).

[0193] In FXR containing cells and in transgenic mice, activation of gene expression by endogenous receptors would create undesirable background levels of reporter protein. To circumvent this potential problem, the DNA binding specificity of VpEcR was altered to mimic that of GR, which binds as a homodimer to an inverted repeat of the sequence AGAACA, spaced by three nucleotides. This altered binding specificity was achieve...

example 3

Assay for Ecdysone Responsiveness in Stable Cell Lines

[0194] Stable cell lines were generated containing the modified ecdysone receptor VpEcR, a heterodimeric partner (RXR), and an ecdysone inducible reporter (FIG. 2). 293 cells were transfected with the following linearized plasmids, pRC-ESHβ, EcREx5-ΔMTV-Luc, CMX-VpEcR, and CMX-hRXRa. The following day, the cells were split 1:10 and were allowed to recover one day prior to selection with 1 mg / ml G418 (GIBCO). After 14 days of selection, 14 individual clones were isolated and grown separately in the presence of 0.5 mg / ml G418. Of 14 G418 resistant clones, 10 demonstrated differing degrees of muristerone responsiveness.

[0195] One of these cell lines, N13, was grown in the presence or absence of 1 μM muristerone for 20 hours. Cell lysates were then assayed for β-galactosidase and luciferase activities as described in Example 1. X-gal staining was performed on the stable cell lines. Cells were fixed briefly with 10% formaldehyde in ...

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Abstract

In accordance with the present invention, there are provided various methods for modulating the expression of an exogenous gene in a mammalian subject employing modified ecdysone receptors. Also provided are modified ecdysone receptors, as well as homomeric and heterodimeric receptors containing sane, nucleic acids encoding invention modified ecdysone receptors, modified ecdysone response elements, gene transfer vectors, recombinant cells, and transgenic animals containing nucleic acids encoding invention modified ecdysone receptor.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. Ser. No. 08 / 974,530, filed Nov. 19, 1997, now pending, which is, in turn, a continuation-in-part of U.S. Ser. No. 08 / 628,830, filed Apr. 5, 1996, now pending, the entire contents of both of which are hereby incorporated by reference herein.FIELD OF THE INVENTION [0002] The present invention relates to methods in the field of recombinant DNA technology, and products related thereto. More particularly, the invention relates to methods and products for modulating the expression of exogenous genes in mammalian systems. BACKGROUND OF THE INVENTION [0003] The steroid / thyroid hormone receptors comprise a superfamily of ligand-dependent transcription factors that play a crucial role in mediating changes in cell fate and function (Evans, R. M., Science 240:889-895 (1988)). The receptors transduce extracellular hormonal signals to target genes that contain specific enhancer sequences referred to as hormone response...

Claims

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Application Information

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IPC IPC(8): A61K48/00A01K67/027C07K14/72C12N15/85
CPCA01K67/0275A01K2217/05A61K48/0058A61K48/0066C12N2830/75C12N15/63C12N15/85C12N2830/002C12N2830/15C07K14/721
Inventor EVANS, RONALDNO, DAVIDSAEZ, ENRIQUE
Owner EVANS RONALD
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