Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-07-10
2001-10-09
Eyler, Yvonne (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S320100, C435S325000, C435S252300, C435S173300, C536S023100, C536S023400
Reexamination Certificate
active
06300488
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods in the field of recombinant DNA technology, and products related thereto. In a particular aspect, the invention relates to methods for modulating the expression of exogenous genes in mammalian systems, and products useful therefor.
BACKGROUND OF THE INVENTION
In the field of genetic engineering, precise control of gene expression is an invaluable tool for studying, manipulating and controlling development and other physiological processes. For example applications for regulated gene expression in mammalian systems include inducible gene targeting, overexpression of toxic and teratogenic genes, anti-sense RNA expression, and gene therapy (see, for example, Jaenisch, R. (1988)
Science
240, 1468-1474). For cultured cells, glucocorticoids and other steroids have been used to induce the expression of a desired gene.
As another means for controlling gene expression in mammalian systems, an inducible tetracycline regulated system has been devised and utilized in transgenic mice, whereby gene activity is induced in the absence of the antibiotic and repressed in its presence (see, e.g, Gossen et al. (1992)
PNAS
89, 5547-5551; Gossen et al.(1993)
TIBS
18, 471-475; Furth et al. (1994)
PNAS
91 9302-9306; and Shockett et al. (1995)
PNAS
92, 6522-6526). However, disadvantages of the inducible tetracycline system include the requirement for continuous administration of tetracycline to repress expression and the slow clearance of antibiotic from bone, which interferes with regulation of gene expression. 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 (see, e.g., Gossen et al. (1995)
Science
268, 1766-1769).
Accordingly, there is a need in the art for improved systems to precisely modulate the expression of exogenous genes in mammalian subjects. For example, a non-mammalian-based transcription regulating system would be extremely desirable for general application to transgene regulation in in vitro, ex vivo, and in vivo applications, as well as transgenic animals. A system that is simple, compact and dependent on ligands which are relatively inexpensive, readily available and of low toxicity in animals would prove useful for stimulation of regulated systems.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, it has been discovered that nuclear receptor proteins isolated from the silk moth
Bombyx mori
(bR) are useful for the regulation of transgene expression. bR is generally thought to be a strong transcriptional regulator within cells of the silk moth. In accordance with the present invention, it has been discovered that bR is also functional in mammalian cells. It has further been discovered that the addition of activation domains to the bR open-reading frame (VbR) enhances the activity of the ligand modulated regulator to afford high-level transcriptional induction (see, e.g., FIG.
1
A). Further modifications to the bR ligand binding domain result in receptors with unique transactivation characteristics (see, e.g., FIG.
1
B).
In accordance with another aspect of the present invention, hybrid proteins produced by fusion of modified bRs with other ligand-regulated proteins have been found to be capable of high level, regulated transactivation of sequences controlled by both hormone response elements and tetracycline operators. VbR variants and hybrid proteins (see, e.g., FIG.
1
C), in combination with the appropriate promoters and transgenes, can be introduced into target cells by common methods such as transfection of plasmids or by virus mediated gene transfer. The small size and simplicity of these proteins makes them particularly attractive for use in retroviral vectors.
Invention methods provide for regulated gene expression by exogenous non-mammalian inducers, and therefor can be advantageously employed in a variety of in vivo and in vitro mammalian expression systems. For example, inducible expression of cre recombinase in transgenic mammals, in accordance with invention methods, would enable those of skill in the art to accomplish temporally specific inducible gene targeting of the adult or the developing embryo (see, for example, O'Gorman et al. (1991)
Science
251, 1351-1355).
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Gage Fred H.
Suhr Steven T.
Basi Nirman S.
Eyler Yvonne
Foley & Lardner
Reiter Stephen E.
The Salk Institute for Biological Studies
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