BASF AG patents

This company has a large portfolio of granted patents and patent applications related to tetracycline-regulated promoters. Patents have been granted in Australia (1), Canada (1) and in the United States (16). Additional applications have been filed in Australia, Canada, China and Europe. The inventions cover different versions of Tet-activating systems, transgenic organisms transformed with tetracycline-regulated promoters including eukaryotes in general, and plants and animals in particular.

Detailed information on the BASF AG patents is provided in the following table.  For more information, please contact BASF AG .

Patent Number

Title, Independent Claims and Summary of Claims

Assignee
US 5464758

  • Earliest priority – 14 June 1993
  • Filed – 14 June 1993
  • Granted – 7 November 1995
  • Expected expiry – 14 June 2013

Title – Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters

Claim 1
A polynucleotide molecule coding for a transactivator fusion protein comprising a prokaryotic tet repressor and a eucaryotic transcriptional activator protein domain.
Claim 10
A polynucleotide molecule coding for a protein, wherein said polynucleotide is operably linked to a minimal promoter and at least one tet operator sequence.
Claim 16
A eucaryotic cell transfected with
(a) a first polynucleotide molecule coding for a transactivator fusion protein comprising a prokaryotic tet repressor and a eucaryotic transcriptional activator protein domain; and
(b) a second polynucleotide molecule coding for a protein, wherein said second polynucleotide molecule is operably linked to a minimal promoter and at least one tet operator sequence.
Claim 18
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 coding for a transactivator fusion protein comprising a prokaryotic tet repressor and a eucaryotic transcriptional activator protein domain, and
a second container means contains a second polynucleotide molecule comprising a minimal promoter operably linked to at least one tet operator sequence, wherein said minimal promoter is capable of being ligated to a heterologous gene sequence coding for a polypeptide.
Claim 19
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 coding for a transactivator fusion protein comprising a prokaryotic tet repressor and a eucaryotic transcriptional activator protein domain, and
a second container means contains a second polynucleotide molecule comprising a minimal promoter operably linked to at least one tet operator sequence, wherein said minimal promoter is capable of being ligated to a heterologous gene sequence coding for a polypeptide.

BASF AG

US 5650298

  • Earliest priority – 14 June 1993
  • Filed – 14 June 1994
  • Granted – 22 July 1997
  • Expected expiry – 22 July 2014
Title – Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters

Claim 1
An isolated DNA molecule for integrating a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA) at a predetermined location in a second target DNA molecule, the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells, the DNA molecule comprising a polynucleotide sequence encoding the tTA flanked at 5′ and 3′ ends by additional polynucleotide sequences of sufficient length for homologous recombination between the DNA molecule and the second target DNA molecule at a predetermined location.
Claim 11
An isolated DNA molecule for integrating a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA) and a tTA-responsive promoter within a predetermined gene of interest in a second target DNA molecule, the DNA molecule comprising:

a) a first polynucleotide sequence comprising a 5′ flanking regulatory region of the gene of interest, operably linked to:

b) a second polynucleotide sequence encoding a tTA, the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and

c) a third polynucleotide sequence comprising a tTA-responsive promoter, operably linked to:

d) a fourth polynucleotide sequence comprising at least a portion of a coding region of the gene of interest;

wherein the first and fourth polynucleotide sequences are of sufficient length for homologous recombination between the DNA molecule and the gene of interest in the second target DNA molecule 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.

Claim 45
A method for producing a host cell having a DNA molecule encoding a tetracycline-controllable transactivator (tTA) integrated at a predetermined location in a second target DNA molecule within the cell, comprising:

a) introducing the DNA molecule of claim 1 into a population of cells under conditions suitable for homologous recombination between the DNA encoding the tTA and the second target DNA molecule; and

b) selecting a cell in which the DNA encoding the tTA has integrated at a predetermined location within the second target DNA molecule.

AU 684524 B2

  • Earliest priority – 14 June 1993
  • Filed – 14 June 1994
  • Granted – 18 December 1997
  • Expected expiry – 14 June 2014
Title – Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters

Claim 1
The same as Claim 1 of US 5650298.
Claim 11
The same as Claim 11 of US 5650298.
Claim 42
A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells.
Claim 52A non-human transgenic animal having a transgene comprising a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a prokaryotic 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.
Claim 55A 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.
CA 2165162

  • Earliest priority – 14 June 1993
  • Filed – 14 June 1994
  • Granted – 23 May 2000
  • Expected expiry – 14 June 2014
Title – Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters

Claim 1
The same as Claim 1 of US 5650298.
Claim 11
The same as Claim 11 of US 5650298.
Claim 42
Use of tetracycline or a tetracycline analogue for the inhibition of a second transgene in a transgenic animal, said animal having
a first transgene comprising a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and
the second transgene comprising a gene of interest operably linked to a tTA-responsive promoter.
Claim 43
The use of tetracycline or a tetracycline analogue for the inhibition of transcription of a second transgene in a transgenic animal, said animal having
a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells, wherein the first transgene is integrated by homologous rrecombination at a predetermined location within a chromosome within cells of the animal; and
the second transgene comprising a gene of interest operably linked to a tTA-responsive promoter.
Claim 44
Use of tetracycline or a tetracycline analogue for inhibiting transcription of the gene of interest in a transgenic animal having a transgenecomprising
a polynucleotide sequence encoding a tetracycline-controllable transactivator (tTA) and
a tTA-responsive promoter,
wherein the transgene is integrated by homologous rrecombination 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.
Claim 48
A method for producing a non-human transgenic animal comprising:
a) introducing a DNA molecule encoding the tTA into a fertilized oocyte;

b) implanting the fertilized oocyte in a pseudopregnant foster mother; and

c) allowing the fertilized oocyte to develop into the non-human transgenic animal to thereby produce the non-human transgenic animal.

Remarks

The related European patent application (EP 705334 A2) is pending.
US 5654168

  • Earliest priority – 14 June 1993
  • Filed – 15 July 1994
  • Granted – 5 August 1997
  • Expected expiry – 5 August 2014
Title – Tetracycline-inducible transcriptional activator and tetracycline-regulated transcription unit

Claim 1
An isolated nucleic acid 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.
Claim 26
A kit comprising a carrier means having in close confinement therein at least two container means comprising:

a) a first container means containing a first nucleic acid encoding a fusion protein which activates transcription, the fusion protein comprising a polypeptide which binds to a first class of tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a polypeptide which activates transcription in enkaryotic cells; and

b) a second container means containing a second nucleic acid comprising a first cloning site for introduction of a first nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence of a first class type.

US 5789156

  • Earliest priority – 14 June 1993
  • Filed – 3 February 1995
  • Granted – 4 August 1998
  • Expected expiry – 4 August 2015
Title – Tetracycline-regulated transcriptional inhibitors

Claim 1
An isolated nucleic acid encoding a fusion protein which inhibits transcription in eukaryotic cells, the fusion protein comprising a first polypeptide which binds to tet operator sequences, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.
Claim 46
A kit comprising a carrier means having in close confinement therein at least two container means comprising:

a) a first container means containing a first nucleic acid encoding a fusion protein which inhibits transcription in eukaryotic cells, the fusion protein comprising a first polypeptide which binds to tet operator sequences either

(I) in the presence but not the absence of tetracycline or a tetracycline analogue or

(ii) in the absence but not the presence of tetracycline or a tetracycline analogue, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells, or a eukaryotic cell line into which said first nucleic acid has been stably introduced; and

b) a second container means containing a second nucleic acid comprising a cloning site for introduction of a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

This patent is a Continuation in part of US 5654168.

US 5589362

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted – 31 December 1996
  • Expected expiry – 31 December 2013
Title – Tetracycline-regulated transcriptional modulators with altered DNA binding specificities

Claim 1
An isolated nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion protein comprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position +4 or +6, operatively linked to a polypeptide which regulates transcription in eukaryotic cells.
Claim 13
A method for regulating transcription of a tet operator-linked gene in an isolated cell, comprising:

introducing into the isolated cell a nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion proteincomprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position 14 or 16, operatively linked to a polypeptide which regulates transcription in eukaryotic cells; and

modulating the concentration of a tetracycline, or analogue thereof, in contact with the isolated cell.

This patent is a Continuation in part of US 5789156.

US 5814618

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted – 29 September 1998
  • Expected expiry – 29 September 2015
Title – Methods for regulating gene expression

Claim 1
A method for regulating expression of a tet operator-linked gene in a cell of a subject, comprising:

introducing into the cell a nucleic acid molecule 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; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject.

Claim 17
A method for regulating expression of a gene in a cell of a subject, comprising:

obtaining the cell from the subject;

introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence;

introducing into the cell a second nucleic acid molecule encoding a fusion protein which inhibits transcription, the fusion proteincomprising a first polypeptide which binds to a tet operator sequence, operatively linked to a second polypeptide which inhibits transcription in eukaryotic cells, to form a modified cell;

administering the modified cell to the subject; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject.

This patent is a Continuation in part of US 5789156.

US 5888981

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted – 30 March 1999
  • Expected expiry – 30 March 2016
Title – Methods for regulating gene expression

Claim 1
A method for regulating expression of a tet operator-linked gene in a cell of a subject, comprising:

introducing into the cell a nucleic acid molecule 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; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject.

Claim 10
A method for regulating expression of a gene in a cell of a subject, comprising:

obtaining the cell from the subject;

introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence;

introducing into the cell a second nucleic acid molecule 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, to form a modified cell;

administering the modified cell to the subject; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject.

This patent is a Continuation in part of US 5650298.

US 6004941

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted – 21 December 1999
  • Expected expiry – 21 December 2016
Title – Methods for regulating gene expression

Claim 1
A method for regulating expression of a tet operator-linked gene in a cell of a subject, comprising:

introducing into the cell a nucleic acid molecule 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; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject, such that expression of a tet operator-linked gene in a cell of the subject is regulated.

Claim 16
A method for regulating expression of a gene in a cell of a subject, comprising:

obtaining the cell from the subject;

introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence;

introducing into the cell a second nucleic acid molecule encoding a fusion protein which activates transcription, the fusion proteincomprising 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, to form a modified cell;

administering the modified cell to the subject; and

modulating the concentration of a tetracycline, or analogue thereof, in the subject such that expression of the gene which is operatively linked to at least one tet operator sequence is regulated in a cell of the subject.

Claim 28
An isolated recombinant vector for coordinate, bidirectional transcription of a first and a second nucleotide sequence, the vector comprising a nucleotide sequence comprising in a 5′ to 3′ direction:

a) a first cloning site for introduction of a first nucleotide sequence to be transcribed, operatively linked to

b) at least one tet operator sequence, operatively linked to

c) a second cloning site for introduction of a second nucleotide sequence transcribed,

the vector further comprising additional regulatory sequences such that the vector is sufficient for use in eukaryotic cells, wherein transcription of a first and second nucleotide sequence introduced into the vector proceeds in opposite directions relative to the at least one tet operator sequence.

Claim 35
An isolated nucleic acid composition comprising at least one recombinant vector for independent regulation of transcription of a first and a second nucleotide sequence, the nucleic acid composition comprising nucleotide sequences comprising:

a) a first cloning site for introduction of a first nucleotide sequence to be transcribed, operatively linked to at least one let operator sequence of a first class type; and

b) a second cloning site for introduction of a second nucleotide sequence to be transcribed, operatively linked to at least one let operator sequence of a second class type.

This patent is a Continuation in part of US 5789156.

US 5859310

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted – 12 January 1999
  • Expected expiry – 12 January 2016
Title – Mice transgenic for a tetracycline-controlled transcriptional activator

Claim 1
A transgenic mouse having a transgene integrated into the genome of the mouse and also having a tet operator-linked gene in the genome of the mouse, wherein:

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a tetracycline-controllable transactivator fusion protein (tTA),

said fusion protein comprises a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription of said tet operator-linked gene in eucaryotic cells,

said tet operator-linked gene confers a detectable and functional phenotype on the mouse when expressed in cells of the mouse,

said transgene is expressed in cells of the mouse at a level sufficient to produce amounts of said fission protein that are sufficient to activate transcription of the tet operator-linked gene; and

in the absence of tetracycline or a tetracycline analogue in the mouse, said fusion protein binds to the tet operator-linked gene and activates transcription of the tet operator linked gene such that the tet operator-linked gene is expressed at a level sufficient to confer the detectable and functional phenotype on the mouse, wherein the level of expression of tet operator-linked gene can be downmodulated by administering tetracycline or a tetracycline analogue to the mouse.

Claim 13
A transgenic mouse having a transgene integrated into the genome of the mouse, wherein:

the transgene comprises a polynucleotide sequence encoding a tetracycline-controllable transactivator fusion protein (tTA) and a tTA-responsive promoter,

said fusion protein comprises a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription of a gene of interest in eucaryotic cells,

the transgene is integrated by homologous recombination at a predetermined location within a said gene of interest within cells of the mouse such that expression of the fission protein 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,

expression of the gene of interest confers a detectable and functional phenotype on the mouse,

said transgene is expressed in cells of the mouse at a level sufficient to produce amounts of said fusion protein that are sufficient to activate transcription of the gene of interest linked to the tTA-responsive promoter, and

in the absence of tetracycline or a tetracycline analogue in the mouse, said fusion protein binds to the tTA-responsive promoter and activates transcription of the gene of interest such that the gene of interest is expressed at a level sufficient to confer the detectable and functional phenotype on the mouse, wherein the level of expression of the gene of interest can be downmodulated by administering tetracycline or a tetracycline analogue to the mouse.

Claim 20
A transgenic mouse having a transgene integrated into the genome of the mouse, wherein;

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a tetracycline-controllable transactivator fusion protein (tTA),

said fruition protein comprises a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription of a tet operator-linked gene in eucaryotic cells, and

said fusion protein is expressed in cells of the mouse.

This patent is a Continuation in part of US 5650298.

US 5866755

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted – 2 February 1999
  • Expected expiry – 2 February 2016
Title – Animals transgenic for a tetracycline-regulated transcriptional inhibitor

Claim 1
A transgenic mouse having a transgene integrated into the genome of the mouse and also having a tet operator-linked gene in the genome of the mouse, wherein:

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of said tet operator linked gene,

said fusion protein comprises a first polypeptide that is a Tet repressor operably linked to a heterologous second polypeptide which inhibits transcription of said tet operator-linked gene in eucaryotic cells,

said tet operator-linked gene confers a detectable and functional phenotype on the mouse when expressed in cells of the mouse,

said transgene is expressed in cells of the mouse at a level sufficient to produce amounts of said fusion protein that are sufficient to inhibit transcription of the tet operator-linked gene; and

in the absence of tetracycline or a tetracycline analogue in the mouse, said fusion protein binds to the tet operator-linked gene and inhibits transcription of the tet operator linked gene, wherein the level of expression of the tet operator-linked gene can be upregulated by administering tetracycline or a tetracycline analogue to the mouse.

Claim 2
A transgenic mouse having a transgene integrated into the genome of the mouse and also having a tet operator-linked gene in the genome of the mouse, wherein:

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of said tet operator linked gene,

said fusion protein comprises a first polypeptide that is a mutated Tet repressor that binds to tet operator sequences in the presence, but not the absence, of tetracycline or a tetracycline analogue, operably linked to a heterologous second polypeptide which inhibits transcription of said tet operator-linked gene in eucaryotic cells,

said tet operator-linked gene confers a delectable and functional phenotype on the mouse when expressed in cells of the mouse,

said transgene is expressed in cells of the mouse at a level sufficient to produce amounts of said fusion protein that are sufficient to inhibit transcription of the ret operator-linked gene; and

in the presence of tetracycline or a tetracycline analogue in the mouse, said fusion protein binds to the tet operator-linked gene and inhibits transcription of the tet operator linked gene, wherein the level of expression of the tet operator-linked gene can be upregulated by depleting tetracycline or a tetracycline analogue from the mouse.

Claim 3
A transgenic mouse having a transgene integrated into the genome of the mouse, wherein:

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of a tet operator linked gene,

the fusion protein comprising a first polypeptide that is a Tet repressor or, a mutated Tet repressor that binds to a tet operator sequence, operatively linked to a second polypeptide which inhibits transcription in eukaryotic cells, and

said fusion protein is expressed in cells of the mouse.

This patent is a Continuation in part of US 5789156.

US 5912411

  • Earliest priority – 14 June 1993
  • Filed – 7 June 1995
  • Granted -15 June 1999
  • Expected expiry – 15 June 2016
Title – Mice transgenic for a tetracycline-inducible transcriptional activator

Claim 1
A transgenic mouse having a transgene integrated into the genome of the mouse and also having a tet operator-linked gene in the genome of the mouse, wherein:

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a fusion protein which activates transcription of said tet operator-linked gene,

the fusion protein comprises a first polypeptide which is a mutated Tet repressor that 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,

said tet operator-linked gene confers a detectable and functional phenotype on the mouse when expressed in cells of the mouse,

said transgene is expressed in cells of the mouse at a level sufficient to produce amounts of said fusion protein that are sufficient to activate transcription of the tet operator-linked gene; and

in the presence of tetracycline or a tetracycline analogue in the mouse, said fusion protein binds to the tet operator-linked gene and activates transcription of the tet operator linked gene such that the tet operator-linked gene is expressed at a level sufficient to confer the detectable and functional phenotype on the mouse, wherein the level of expression of the tet operator-linked gene cap be downmodulated by depleting tetracycline or a tetracycline analogue from the mouse.

Claim 35 
A transgenic mouse having a transgene integrated into the genome of the mouse, wherein:

the transgene comprises a transcriptional regulatory element functional in cells of the mouse operatively linked to a polynucleotide sequence encoding a fusion protein which activates transcription of a tet operator-linked gene,

the fusion protein comprising a first polypeptide which is a mutated Tet repressor that 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, and

said fusion protein is expressed, in cells of the mouse.

This patent is a Continuation in part of US 5789156.

US 5922927

  • Earliest priority – 14 June 1993
  • Filed – 21 July 1997
  • Granted -13 July 1999
  • Expected expiry – 14 June 2013
Title – Methods for producing tetracycline-regulated transgenic mice

Claim 1
A method for producing a transgenic mouse, comprising:

a) introducing into a fertilized oocyte of a mouse a DNA molecule encoding a tetracycline-controllable transactivator (tTA), the tTAcomprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells;

b) implanting the fertilized oocyte in a pseudopregnant foster mother; and

c) allowing the fertilized oocyte to develop into a transgenic mouse to thereby produce the transgenic mouse, wherein said tTA is expressed in cells of the mouse at a level sufficient to transactivate a tet operator-linked gene.

Claim 2
A method for producing a transgenic mouse having a transgene encoding a tetracycline-controllable transactivator (tTA) integrated at a predetermined location within chromosomal DNA of cells of the mouse, comprising:

a) introducing into a population of embryonic stem cells of a mouse a DNA molecule encoding a tTA, the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells, the DNA molecule comprising a polynucleotide sequence encoding the tTA flanked at 5′ and 3′ ends by additional polynucleotide sequences of sufficient length for homologous recombination between the DNA molecule and a second target DNA molecule at a predetermined location within chromosomal DNA of cells of the mouse, under conditions suitable for homologous recombination between the DNA molecule encoding the tTA and chromosomal DNA within the cell;

b) selecting an embryonic stem cell in which DNA encoding the tTA has integrated at a predetermined location within the chromosomal DNA of the cell;

c) implanting the embryonic stem cell into a blastocyst;

d) implanting the blastocyst into a pseudopregnant foster mother; and

e) allowing the blastocyst to develop into a transgenic mouse to thereby produce the transgenic mouse, wherein said tTA is expressed in cells of the mouse at a level sufficient to transactivate a tet operator-linked gene.

Claim 3
A method for producing a transgenic mouse having a transgene encoding a tetracycline-controllable transactivator (tTA) and a tTA-responsive promoter integrated at a predetermined location within a gene of interest in cells of the mouse, comprising:

a) introducing into a population of embryonic stem cells of a mouse a DNA molecule encoding a tTA, the DNA molecule comprising:

i) a first polynucleotide sequence comprising a 5′ flanking regulatory region of the gene of interest, operably linked to:

ii) a second polynucleotide sequence encoding a tTA, the tTA comprising a prokaryotic Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and

iii) a third polynucleotide sequence comprising a tTA-responsive promoter, operably linked to:

iv) a fourth polynucleotide sequence comprising at least a portion of a coding region of the gene of interest;

wherein the first and fourth polynucleotide sequences are of sufficient length for homologous recombination between the DNA molecule and the gene of interest 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, under conditions suitable for homologous recombination between the DNA molecule encoding the tTA and the gene of interest within the cell;

b) selecting an embryonic stem cell in which DNA encoding the tTA has integrated at a predetermined location within the gene of interest in the cell;

c) implanting the embryonic stem cell into a blastocyst;

d) implanting the blastocyst into a pseudopregnant foster mother; and

e) allowing the blastocyst to develop into a transgenic mouse to thereby produce the transgenic mouse,

wherein said gene of interest confers a detectable and functional phenotype on the mouse when expressed in cells of the transgenic mouse,

said tTA is expressed in cells of the transgenic mouse at a level sufficient to activate transcription of the gene of interest; and

in the absence of tetracycline or a tetracycline analogue in the mouse, said tTA binds to the tTA responsive promoter operably linked to the gene of interest and activates transcription of the gene of interest such that the gene of interest is expressed at a level sufficient to confer the detectable and functional phenotype on the mouse, wherein the level of expression of the gene of interest can be downmodulated by administering tetracycline or a tetracycline analogue to the mouse.

This patent is a Division of US 5650298.

US 6136954

  • Earliest priority – 14 June 1993
  • Filed – 28 september 1998
  • Granted – 24 October 2000
  • Expected expiry – 14 June 2013
Title – Tetracycline-inducible transcriptional activator fusion proteins

Claim 1
A fusion protein which activates transcription comprising a first polypeptide which binds to a test operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.

This patent is a Continuation of US 5814618.

US 6242667

  • Earliest priority – 14 June 1993
  • Filed – 28 september 1998
  • Granted – 5 June 2001
  • Expected expiry – 14 June 2013
Title – Transgenic organisms having tetracycline-regulated transcriptional regulatory systems

Claim 1

A transgenic plant having a transgene integrated into the genome of the plant and also having a tet operator-linked gene in the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which activates transcription of said tet operator linked gene,
the fusion protein comprises a first polypeptide which is a mutated Tet repressor that 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,
said tet operator-linked gene confers a detectable and functional phenotype on the plant when expressed in cells of the plant,
said transgene is expressed in cells of the plant at a level sufficient to produce amounts of said fusion protein that are sufficient to activate transcription of the tet operator-linked gene; and
in the presence of tetracycline or a tetracycline analogue in the plant, said fusion protein binds to the tet operator-linked gene and activates transcription of the tet operator linked gene such that the tet operator-linked gene is expressed at a level sufficient to confer the detectable and functional phenotype on the plant, wherein the level of expression of the tet operator-linked gene can be downmodulated by depleting tetracycline or a tetracycline analogue from the plant.

Claim 2

A transgenic plant having a transgene integrated into the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which activates transcription of a tet operator linked gene,
the fusion protein comprising a first polypeptide which is a mutated Tet repressor that 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, and
said fusion protein is expressed in cells of the plant.

Claim 3

A transgenic plant having a transgene integrated into the genome of the plant and also having a tet operator-linked gene in the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of said tet operator linked gene,
the fusion protein comprises a first polypeptide which is a mutated Tet repressor that binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which inhibits transcription in eukaryotic cells,
said tet operator-linked gene confers a detectable and functional phenotype on the plant when expressed in cells of the plant,
said transgene is expressed in cells of the plant at a level sufficient to produce amounts of said fusion protein that are sufficient to inhibit transcription of the tet operator-linked gene; and
in the presence of tetracycline or a tetracycline analogue in the plant, said fusion protein binds to the tet operator-linked gene and inhibits transcription of the tet operator linked gene, wherein the level of expression of the tet operator-linked gene can be upregulated by depleting tetracycline or a tetracycline analogue from the plant.

Claim 4

A transgenic plant having a transgene integrated into the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of a tet operator linked gene,
the fusion protein comprising a first polypeptide which is a mutated Tet repressor that binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which inhibits transcription in eukaryotic cells, and
said fusion protein is expressed in cells of the plant.

Claim 5

A transgenic plant having a transgene integrated into the genome of the plant and also having a tet operator-linked gene in the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of said tet operator linked gene,
said fusion protein comprises a first polypeptide that is a Tet repressor, operably linked to a heterologous second polypeptide which inhibits transcription of said tet operator-linked gene in eukaryotic cells,
said tet operator-linked gene confers a detectable and functional phenotype on the plant when expressed in cells of the plant,
said transgene is expressed in cells of the plant at a level sufficient to produce amounts of said fusion protein that are sufficient to inhibit transcription of the tet operator-linked gene; and
in the absence of tetracycline or a tetracycline analogue in the plant, said fusion protein binds to the tet operator-linked gene and inhibits transcription of the tet operator linked gene, wherein the level of expression of the tet operator-linked gene can be upregulated by administering tetracycline or a tetracycline analogue to the plant.

Claim 6

A transgenic plant having a transgene integrated into the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which inhibits transcription of a tet operator linked gene,
the fusion protein comprising a first polypeptide which is a Tet repressor, operatively linked to a second polypeptide which inhibits transcription in eukaryotic cells, and said fusion protein is expressed in cells of the plant.

This patent is a Continuation of US 5912411.

US 6252136

  • Earliest priority – 14 June 1993
  • Filed – 29 september 1998
  • Granted – 26 June 2001
  • Expected expiry – 14 June 2013
Title – Transgenic organisms having tetracycline-regulated transcriptional regulatory systems

Claim 1
A transgenic plant having a transgene integrated into the genome of the plant and also having a tet operator-linked gene in the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which activates transcription of said tet operator linked gene,
the fusion protein comprises a first polypeptide which is a Tet repressor operatively linked to a second polypeptide which directly or indirectly activates transcription in plant cells,
said tet operator-linked gene confers a detectable and functional phenotype on the plant when expressed in cells of the plant,
said transgene is expressed in cells of the plant at a level sufficient to produce amounts of said fusion protein that are sufficient to activate transcription of the tet operator-linked gene; and
in the absence of tetracycline or a tetracycline analogue in the plant, said fusion protein binds to the tet operator-linked gene and activates transcription of the tet operator linked gene such that the tet operator-linked gene is expressed at a level sufficient to confer the detectable and functional phenotype on the plant, wherein the level of expression of the tet operator-linked gene can be downmodulated by administering tetracycline or a tetracycline analogue to the plant.
Claim 7
A transgenic plant having a transgene integrated into the genome of the plant, wherein:
the transgene comprises a transcriptional regulatory element functional in cells of the plant operatively linked to a polynucleotide sequence encoding a fusion protein which activates transcription of a tet operator linked gene,
the fusion protein comprising a first polypeptide which is a Tet repressor, operatively linked to a second polypeptide which directly or indirectly activates transcription in plant cells, and
said fusion protein is expressed in cells of the plant.

This patent is a Continuation of US 5859310.

US 6271348

  • Earliest priority – 14 June 1993
  • Filed – 24 January 2000
  • Granted – 7 August 2001
  • Expected expiry – 14 June 2013
Title – Tetracycline-inducible transcriptional inhibitor fusion proteins

Claim 1

A fusion protein which inhibits transcription in eukaryotic cells, the fusion protein comprising a first polypeptide which binds to tet operator sequences, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.

This patent is a Division of US 6136954.

AU 746850 B2

  • Earliest priority – 29 June 1995
  • Filed – 18 August 1999
  • Granted – 2 May 2002
  • Expected expiry – 18 August 2019
Title – Tetracycline-regulated transcriptional modulaters

Claim 1
An isolated nucleic acid encoding a fusion protein which activates transcription, the fusion protein comprising a first  polypeptide which binds to a tet operator sequence in the presence, but not the absence, of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.
Claim 12
A fusion protein which activates transcription comprising a first polypeptide which binds to a tet operator sequence in the presence, but not the absence, of tetracycline or a tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.
Claim 35
An isolated nucleic acid 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.
Claim 51
A fusion protein which inhibits transcription in eukaryotic cells, comprising a first polypeptide which binds to a let operator sequence operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.
Claim 73
A host cell comprising:
a first nucleic acid encoding a first fusion protein which activates transcription, the first fusion protein comprising a first polypeptide which binds to a tet operator sequence operatively linked to a second polypeptide which activates transcription in eukaryotic cells;
a second nucleic acid encoding a second fusion protein which inhibits transcription, the second fusion protein comprising a third polypeptide which binds to a tet operator sequence operatively linked to a fourth polypeptide which inhibits transcription in eukaryotic cells; and
a third nucleic acid molecule comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.
Claim 76
A non-human transgenic organism comprising:
a first transgene encoding a first fusion protein which activates transcription, the first fusion protein comprising a first polypeptide which binds to a tet operator sequence operatively linked to a second polypeptide which activates transcription in eukaryotic cells;
a second transgene encoding a second fusion protein which inhibits transcription, the second fusion protein comnprising a third polypeptide which binds to a tet operator sequence operatively linked to a fourth polypeptide which inhibits transcription in eukaryotic cells; and
a third transgene comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.
Claim 79
A recombinant vector for coordinate, bidirectional transcription of a first and a second nucleotide sequence to be transcribed, the vectorcomprising a nucleotide sequence comprising in a 5′ to 3′ direction:
a first cloning site for introduction of a first nucleotide sequence to be transcribed, which is operatively linked to at least one tet operator sequence, which is operatively linked to a second cloning site for introduction of a second nucleotide sequence to be transcribed, wherein transcription of the first and second nucleotide sequence introduced into the vector proceeds in opposite directions relative to the at least one tet operator sequence.
Claim 83
A composition of matter comprising at least one recombinant vector for independent regulation of transcription of a first and a second nucleotide sequence to be transcribed, the at least one vector comprising a nucleotide sequence comprising:
a first cloning site for introduction of a first nucleotide sequence to be transcribed, operatively linked to at least one tet operator sequence of a first class type; and
a second cloning site for introduction of a second nucleotide sequence to be transcribed, operatively linked to at least one tet operator sequence of a second class type.
Claim 87
A kit comprising a carrier means having in close confinement therein at least two container means comprising:
a first container means containing a first nucleic acid encoding a fusion protein which activates transcription, the fusion proteincomprising a first polypeptide which binds to a let operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells; and
a second container means containing a second nucleic acid comprising a cloning site for introduction of a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

BASF AG

andAbbott GmbH & Co. KG ( a subsidiary of Abbott Labs)

Remarks

Related patents were granted in Canada (CA 2193122) and Europe ( EP 804565). Related applications are pending in Europe (EP 1092771), China (CN 1167504 A and CN 1523112), Japan (JP 9500526 T2 & JP 11506901 T2) and Norway (NO 9605623 A).

Note: patent infomation was last update on 10 May 2006. Search terms: “BASF” in applicant and “tetracycline” in abstract. Patent database: PatentLens in combination with INPADOC.