Animals transgenic for a tetracycline-regulated transcriptional inhibitor

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 inhibited by a transcriptional inhibitor fuision protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences and a second polypeptide which directly or indirectly inhibits transcription in eukaryotic cells. In various embodiment, the first polypeptide binds to tet operator sequences either: (i) in the absence but not the presence of tetracycline (or an analogue thereof) or (ii) in the presence but not the absence of tetracycline (or an analogue thereof). In a preferred embodiment, the transgene encoding the transcriptional inhibitor 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 polyncleotide sequence encoding a fusion protein which inhibits transcription in eukaryotic cells, 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.  
     
     
         2 . The animal of  claim 1 , wherein the first polypeptide of the fusion protein binds to tet operator sequences in the absence but not the presence of tetracycline or a tetracycline analogue.  
     
     
         3 . The animal of  claim 2 , wherein the first polypeptide is a Tet repressor.  
     
     
         4 . The animal of  claim 3 , wherein the first polypeptide comprises an amino acid sequence shown in SEQ ID NO: 17.  
     
     
         5 . The animal of  claim 1 , wherein the first polypeptide of the fusion protein binds to tet operator sequences in the presence but not the absence of tetracycline or a tetracycline analogue.  
     
     
         6 . The animal of  claim 5 , wherein the first polypeptide is a mutated Tet repressor.  
     
     
         7 . The animal of  claim 6 , wherein the mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor.  
     
     
         8 . The animal of  claim 7 , wherein the mutated Tet repressor has an amino acid substitution at at least one amino acid position corresponding to an amino acid position selected from the group consisting of position 71, position 95, position 101 and position 102 of a wild-type Tn10-derived Tet repressor amino acid sequence.  
     
     
         9 . The animal of  claim 8 , wherein the mutated Tet repressor comprises an amino acid sequence shown in SEQ ID NO: 19.  
     
     
         10 . The animal of  claim 1 , wherein the second polypeptide comprises a transcription silencer domain of a protein from the group consisting of v-erbA, the Drosophila Krueppel dorsal protein, TSF3, SFI, the Drosophila hunchback protein, the Drosophila knirps protein, WT1, Oct-2.1, the Drosophila engrailed protein, E4BP4 and ZF5.  
     
     
         11 . The animal of  claim 1 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         12 . The animal of  claim 1 , which is a mouse.  
     
     
         13 . The animal of  claim 1 , which is selected from a group consisting of a cow, a goat, a sheep and a pig.  
     
     
         14 . A method for modulating transcription of the second transgene in the transgenic animal of  claim 11 , comprising administering tetracycline or a tetracycline analogue to the animal.  
     
     
         15 . A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a fusion protein which inhibits transcription in eukaryotic cells, 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, wherein the transgene is integrated by at a predetermined location within a chromosome within cells of the animal.  
     
     
         16 . The animal of  claim 15 , wherein the first polypeptide of the fusion protein binds to tet operator sequences in the absence but not the presence of tetracycline or a tetracycline analogue.  
     
     
         17 . The animal of  claim 16 , wherein the first polypeptide is a Tet repressor.  
     
     
         18 . The animal of  claim 17 , wherein the first polypeptide comprises an amino acid sequence shown in SEQ ID NO: 17.  
     
     
         19 . The animal of  claim 17 , wherein the first polypeptide of the fusion protein binds to tet operator sequences in the presence but not the absence of tetracycline or a tetracycline analogue.  
     
     
         20 . The animal of  claim 19 , wherein the first polypeptide is a mutated Tet repressor.  
     
     
         21 . The animal of  claim 20 , wherein the mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor.  
     
     
         22 . The animal of  claim 21 , wherein the mutated Tet repressor has an amino acid substitution at at least one amino acid position corresponding to an amino acid position selected from the group consisting of position 71, position 95, position 101 and position 102 of a wild-type Tn10-derived Tet repressor amino acid sequence.  
     
     
         23 . The animal of  claim 22 , wherein the mutated Tet repressor comprises an amino acid sequence shown in SEQ ID NO: 19.  
     
     
         24 . The animal of  claim 15 , wherein the second polypeptide comprises a transcription silencer domain of a protein selected from the group consisiting of v-erbA, the Drosophila Krueppel protein, the retinoic acid receptor alpha, the thyroid hormone receptor alpha, the yeast Ssn6/Tup1 protein complex, the Drosophila protein even-skipped, SIR1, NeP1, the Drosophila dorsal protein, TSF3, SFI, the Drosophila hunchback protein, the Drosophila knirps protein, WT1, Oct-2.1, the Drosophila engrailed protein, E4BP4 and ZF5.  
     
     
         25 . The animal of  claim 15 , further having a second transgene comprising a gene of interest operably linked to at least one tet operator sequence.  
     
     
         26 . A method for modulating transcription of the second transgene in the transgenic animal of  claim 25 , comprising administering tetracycline or a tetracycline analogue to the animal.