Transgenic organisms having tetracycline-regulated transcriptional regulatory systems

Abstract

Transgenic animals carrying a transgene comprising a nucleic acid molecule encoding protein useful for regulating the expression of genes in eukaryotic cells and organisms in a highly controlled manner are disclosed. In the regulatory system of the invention, transcription of a tet operator-linked nucleotide sequence is stimulated by a transcriptional activator fusion protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences in the presence of tetracycline operatively linked to a second polypeptide activates transcription in eukaryotic cells. In a preferred embodiment, the transgene encoding the transcriptional activator fusion protein is integrated at a predetermined location within the chromosome of the transgenic animal.

1 . A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a fusion protein which activates transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.  
     
     
         2 . The animal of  claim 1 , wherein the first polypeptide of the fusion protein is a mutated Tet repressor.  
     
     
         3 . The animal of  claim 2 , wherein the mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor.  
     
     
         4 . The animal of  claim 3 , wherein the mutated Tet repressor is a mutated Tn10-derived Tet repressor having an amino acid substitution at at least one amino acid position selected from the group consisting of position 71, position 95, position 101 and position 102.  
     
     
         5 . The animal of  claim 4 , wherein the mutated Tn10-derived Tet repressor comprises an amino acid sequence shown in positions 1 to 207 of SEQ ID NO: 2.  
     
     
         6 . The animal of  claim 1 , wherein the second polypeptide of the fusion protein comprises a transcription activation domain of herpes simplex virion protein 16.  
     
     
         7 . The animal of  claim 1 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         8 . The animal of  claim 7 , further having a third transgene comprising a polynucleotide sequence encoding a fusion protein which inhibits transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.  
     
     
         9 . The animal of  claim 1 , which is a mouse.  
     
     
         10 . The animal of  claim 1 , which is selected from a group consisting of a cow, a goat, a sheep and a pig.  
     
     
         11 . A method for modulating transcription of the second transgene in the transgenic animal of  claim 7 , comprising administering tetracycline or a tetracycline analogue to the animal.  
     
     
         12 . A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a fusion protein which activates transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells, wherein the transgene is integrated at a predetermined location within a chromosome within cells of the animal.  
     
     
         13 . The animal of  claim 12 , wherein the first-polypeptide of the fusion protein is a mutated Tet repressor.  
     
     
         14 . The animal of  claim 13 , wherein the mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor.  
     
     
         15 . The animal of  claim 14 , wherein the mutated Tet repressor is a mutated Tn10-derived Tet repressor having an amino acid substitution at at least one amino acid position selected from the group consisting of position 71, position 95, position 101 and position 102.  
     
     
         16 . The animal of  claim 15 , wherein the mutated Tn10-derived Tet repressor comprises an amino acid sequence shown in positions 1 to 207 of SEQ ID NO: 2.  
     
     
         17 . The animal of  claim 12 , wherein the second polypeptide of the fusion protein comprises a transcription activation domain of herpes simplex virion protein 16.  
     
     
         18 . The animal of  claim 12 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         19 . The animal of  claim 18 , further having a third transgene comprising a polynucleotide sequence encoding a fusion protein which inhibits transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.  
     
     
         20 . A method for modulating transcription of the second transgene in the transgenic animal of  claim 18 , comprising administering tetracycline or a tetracycline analogue to the animal.