Transgenic organisms having tetracycline-regulated transcriptional regulatory systems

Abstract

Transgenic animals carrying two transgenes, the first coding for a transactivator fusion protein comprising a tet repressor and a polypeptide which directly or indirectly activates in eucaryotic cells, and the second comprising a gene operably linked to a minimal promotor operably linked to at least one tet operator sequence, are disclosed. Isolated DNA molecules (e.g., targeting vectors) for integrating a polynucleotide sequence encoding a transactivator of the invention at a predetermined location within a second target DNA molecule by homologous recombination are also disclosed. Transgenic animals having the DNA molecules of the invention integrated at a predetermined location in a chromosome by homologous recombination are also encompassed by the invention. Methods to regulate the expression of a tet operator linked-gene of interest by administering tetracycline or a tetracycline analogue to an animal of the invention are also disclosed. The regulatory system of the invention allows for conditional inactivation or modulation of expression of a gene of interest in a host cell or animal.

1 . A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells.  
     
     
         2 . The animal of  claim 1 , wherein the Tet repressor of the tTA is a Tn 10-derived Tet repressor.  
     
     
         3 . The animal of  claim 1 , wherein the polypeptide of the tTA which directly or indirectly activates transcription in eucaryotic cells is from herpes simplex virus virion protein 16.  
     
     
         4 . The animal of  claim 1 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         5 . The animal of  claim 1 , which is a mouse.  
     
     
         6 . The animal of  claim 1 , which is selected from a group consisting of a cow, a goat, a sheep and a pig.  
     
     
         7 . The animal of  claim 4 , wherein a disease condition can be induced or inhibited in the animal by administering tetracycline or a tetracycline analogue to the animal.  
     
     
         8 . A method for inhibiting transcription of the second transgene in the transgenic animal of  claim 4 , comprising administering tetracycline or a tetracycline analogue to the animal.  
     
     
         9 . A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells, wherein the transgene is integrated by homologous recombination at a predetermined location within a chromosome within cells of the animal.  
     
     
         10 . The animal of  claim 9 , wherein the Tet repressor of the tTA is a Tn10 -derived Tet repressor.  
     
     
         11 . The animal of  claim 9 , wherein the polypeptide of the tTA which directly or indirectly activates transcription in eucaryotic cells is from herpes simplex virus virion protein 16.  
     
     
         12 . The animal of  claim 9 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         13 . The animal of  claim 9 , which is a mouse.  
     
     
         14 . The animal of  claim 12 , wherein a disease condition can be induced or inhibited in the animal by administering tetracycline or a tetracycline analogue to the animal.  
     
     
         15 . A method for inhibiting transcription of the second transgene in the transgenic animal of  claim 12 , comprising administering tetracycline or a tetracycline analogue to the animal.  
     
     
         16 . A transgenic animal having a transgene comprising a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA) and a tTA-responsive promoter, wherein the transgene is integrated by homologous recombination at a predetermined location within a gene of interest within cells of the animal such that expression of the tTA is controlled by 5′ regulatory elements of the gene of interest and expression of the gene of interest is controlled by the tTA-responsive promoter.  
     
     
         17 . The animal of  claim 16 , wherein the Tet repressor of the tTA is a Tn10-derived Tet repressor.  
     
     
         18 . The animal of  claim 16 , wherein the polypeptide of the tTA which directly or indirectly activates transcription in eucaryotic cells is from herpes simplex virus virion protein 16.  
     
     
         19 . The animal of  claim 16 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         20 . The animal of  claim 16 , which is a mouse.  
     
     
         21 . The animal of  claim 19 , wherein a disease condition can be induced or inhibited in the animal by administering tetracycline or a tetracycline analogue to the animal.  
     
     
         22 . A method for inhibiting transcription of the second transgene in the transgenic animal of  claim 19 , comprising administering tetracycline or a tetracycline analogue to the animal.