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US 5,851,796 AGranted
Autoregulatory tetracycline-regulated system for inducible gene expression in eucaryotes
AssigneeYale University
InventorDavid G. Schatz
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AssigneeYale University
InventorDavid G. Schatz
Estimating the term…
A tetracycline-regulated system which provides autoregulatory, inducible gene expression in cultured cells and transgenic animals is described. In the autoregulatory plasmid pTet-tTAk, a modified tTA gene called tTAk was placed under the control of Tetp. Tetracycline prevents tTA from binding to Tetp, preventing expression of both tTA and luciferase. This negative feedback cycle ensures that little or no tTA is produced in the presence of tetracycline, thereby reducing or eliminating possible toxic effects. When tetracycline is removed, however, this strategy predicts that tiny amounts of tTA protein (which may result from the leakiness of the minimal promoter), will bind to Tet-op and stimulate expression of the tTAk gene. A positive feedforward loop is initiated which in turn leads to higher levels of expression of tTA and thus, luciferase. Polynucleotide molecules encoding the autoregulatory system, as well as methods of enhancing or decreasing the expression of desired genes, and kits for carrying out these methods are described.
A polynucleotide comprising a nucleotide sequence encoding a tetracycline transactivator fusion protein, said protein comprising a prokaryotic tet repressor and a eucaryotic transcriptional activator protein, and said polynucleotide molecule being operably linked to an inducible minimal promoter, which promoter contains at least one tet operator sequence.
A method to inhibit expression of a heterologous protein in a eucaryotic cell comprising
A method to enhance the expression of a heterologous protein in a eucaryotic cell comprising
A method to activate the expression of a heterologous protein in a eucaryotic cell comprising
A kit comprising a carrier means having in close confinement therein at least two container means, wherein a first container means contains a first polynucleotide molecule encoding a tetracycline transactivator fusion protein, said protein comprising a procaryotic tet repressor and a eucaryotic transcriptional activator protein, and said polynucleotide molecule being operably linked to an inducible minimal promoter, which promoter contains at least one tet operator sequence;
A kit comprising a carrier means having in close confinement therein at least two container means, wherein a first container means contains a eucaryotic cell transfected with a first polynucleotide molecule encoding a tetracycline transactivator fusion protein, said protein comprising a procaryotic tet repressor and a eucaryotic transcriptional activator protein, and said polynucleotide molecule being operably linked to an inducible minimal promoter, which promoter contains at least one tet operator sequence;
Dependencies are inferred from the claim text.