AU746850B2

AU746850B2 - Tetracycline-regulated transcriptional modulaters

Info

Publication number: AU746850B2
Application number: AU44566/99A
Authority: AU
Inventors: Hermann Bujard; Manfred Gossen
Original assignee: BASF SE; Abbott GmbH and Co KG
Current assignee: TET Systems Holding GmbH and Co KG
Priority date: 1994-07-01
Filing date: 1999-08-18
Publication date: 2002-05-02
Anticipated expiration: 2015-06-29
Also published as: AU4456699A

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

A~boI4 GmId4 Cc, kG Name of Applicant/s: Actual Inventor/s: Address for Service: Invention Title: AbboH- Gmb A Co ke Basf Aktiengesellschaft and Knell Akti.ngIcollihafl Hermann Bujard and Manfred Gossen BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 'TETRACYCLINE-REGULATED TRANSCRIPTIONAL

MODULATERS'

Details of Original Application No. 30923/95 dated 29 JUN 1995 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 22670.00 la TETRACYCLINE-REGULATED TRANSCRIPTIONAL MODULATORS Background of the Invention Functional analysis of cellular proteins is greatly facilitated through changes in the expression level of the corresponding gene for subsequent analysis of the accompanying phenotype. For this approach, an inducible expression system controlled by an external stimulus is desirable. Ideally such a system would not only mediate an "on/off" status for gene expression but would also permit limited expression of a gene at a defined level.

Attempts to control gene activity have been made using various inducible eukaryotic promoters, such as those responsive to heavy metal ions (Mayo et al. (1982) Cell 22:99-108; Brinster et al. (1982) Nature 226:39-42; Searle et al. (1985) Mol. Cell. Biol. 5:1480-1489), heat shock (Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L. CRC, Boca Raton, FL, pp167-220) or hormones (Lee et al. (1981) Nature 294:228-232; Hynes et al. (1981) Proc. Natl. Acad Sci. USA 28:2038-2042; Klock et al. (1987) Nature 322:734-736; Israel Kaufman (1989) Nucl. Acids Res. 12:2589-2604). However, these systems have generally suffered from one or both of the following problems: the inducer heavy metal ions, heat shock or steroid hormones) evokes pleiotropic effects, which can complicate analyses, and many promoter systems exhibit high levels of basal activity in the non-induced state, which prevents shut-off the regulated gene and results in modest induction factors.

An approach to circumventing these limitations is to introduce regulatory elements from evolutionarily distant species such as E.coli into higher eukaryotic cells with the anticipation that effectors which modulate such regulatory circuits will be inert to eukaryotic cellular physiology and, consequently, will not elicit pleiotropic effects in eukaryotic cells.

For example, the Lac repressor (lacR)/operator/inducer system ofE. coli functions in 25 eukaryotic cells and has been used to regulate gene expression by three different approaches: prevention of transcription initiation by properly placed lac operators at promoter sites (Hu Davidson (1987) Cell 48:555-566; Brown et al. (1987) Cell 42:603-612; Figge et al.

(1988) Cell 52:713-722; Fuerst et al. (1989) Proc. Natl. Acad Sci. USA 86:2549-2553: Deuschle et al. (1989) Proc. Natl. Acad Sci. USA 86:5400-5405); blockage of transcribing RNA polymerase II during elongation by a LacR/operator complex (Deuschle et al. (1990) Science 248:480-483); and activation of a promoter responsive to a fusion between LacR and the activation domain of herpes simples virus (HSV) virion protein 16 (VP16) (Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Bairn et al. (1991) Proc. Natl.

Acad Sci. USA 88:5072-5076).

In one version of the Lac system, expression of lac operator-linked sequences is constitutively activated by a LacR-VP 16 fusion protein and is turned off in the presence of isopropyl-p-D-thiogalactopyranoside (IPTG) (Labow et al. (1990), cited supra). In another version of the system, a lacR-VP16 variant is used which binds to lac operators in the presence of IPTG, which can be enhanced by increasing the temperature of the cells (Baim et -2al. (1991), cited supra). The utility of these lac systems in eukaryotic cells is limited, in part, because IPTG acts slowly and inefficiently in eukaryotic cells and must be used at concentrations which approach cytotoxic levels. Alternatively, use of a temperature shift to induce gene expression is likely to elicit pleiotropic effects in the cells. Thus, there is a need for a more efficient inducible regulatory system which exhibits rapid and high level induction of gene expression and in which the inducer is tolerated by eukaryotic cells without cytoxicity or pleiotropic effects.

Components of the tetracycline (Tc) resistance system ofE. coli have also been found to function in eukaryotic cells and have been used to regulate gene expression.

For example, the Tet repressor (TetR), which binds to tet operator sequences in the absence of tetracycline and represses gene transcription, has been expressed in plant cells at sufficiently high concentrations to repress transcription from a promoter containing tet :operator sequences (Gatz, C. et al. (1992) Plant J. 2:397-404). However, very high intracellular concentrations of TetR are necessary to keep gene expression down- 15 regulated in cells, which may not be achievable in many situations, thus leading to *"leakiness" in the system.

In other studies, TetR has been fused to the activation domain of VP16 to create a tetracycline-controlled transcriptional activator (tTA) (Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). The tTA fusion protein is regulated by tetracycline in the same manner as TetR, tTA binds to tet operator sequences in the absence of tetracycline but not in the presence of tetracycline. Thus, in this system, in the continuous presence of Tc, gene expression is kept off, and to induce transcription, Tc is removed.

-3- Summary of the Invention The present invention pertains to an inducible regulatory system which utilizes components of the Tet repressor/operator/inducer system of prokaryotes to stimulate gene expression in eukaryotic cells. In the system of the invention, transcription of a tet operator-linked nucleotide sequence is kept silent in the absence of tetracycline but can be rapidly and strongly induced in the presence of tetracycline (or analogue thereof).

Transcription is induced by a fusion protein composed of at least two polypeptides, a first polypeptide which binds to tet operator sequences in the presence of tetracycline or a tetracycline analogue and a second polypeptide which directly or indirectly activates transcription in eukaryotic cells. In a preferred embodiment, the first polypeptide of the :.fusion protein is a mutated Tet repressor which is regulated by Tc in a reverse manner from the wild type repressor, it binds to tet operator sequences in the presence rather l: than in the absence of Tc. Thus, in the absence of the inducing agent (Tc or a Tc analogue), transcription of a tet operator-linked nucleotide sequence remains uninduced.

S. 15 In the presence of the inducing agent, transcription of the tet operator-linked nucleotide sequence is stimulated by the transactivator fusion protein of the invention.

The inducible regulatory system of the invention has the advantageous properties that induction of gene expression is rapid, efficient and strong typically between 1000 to 2000-fold; up to a 20,000-fold increase in expression has been observed) and the inducing agent does not have to be continuously present. Moreover, the inducing agent does not cause pleiotropic effects or cytotoxicity in eukaryotic cells. The inducible regulatory system of the invention can be applied to regulation of gene expression in cells either in vitro or in vivo, and may be particularly useful for gene therapy applications and for expression of gene products in transgenic and homologous recombinant organisms animals and plants).

-4- The inducible regulatory system of the invention involves at least two components: a tetracycline-inducible transcriptional activator and a target transcription unit to be regulated. Accordingly, the invention further relates to a Tc-inducible transcriptional activator ("transactivator") fusion protein, and nucleic acid DNA) encoding the fusion protein. A preferred fusion protein comprises a Tnl0-encoded Tet repressor that is mutated at at least one amino acid position selected from amino acid positions 71, 101 and/or 102, operatively linked to a herpes simplex virus virion protein 16 (VP16) activation domain (the nucleotide and amino acid sequences of such a fusion protein are shown in SEQ ID NO: 1 and 2, respectively). In one embodiment, the activation domain of the fusion protein includes about 127 C-terminal amino acids of VP16 the fusion protein of SEQ ID NO: In another embodiment, the activation domain includes at least one copy of about 11 C-terminal amino acids of VP16 the amino acid sequence of which is shown in SEQ ID NO:4). Other mutated Tet repressors and transcriptional activation domains having the requisite functional activities are within the scope of the invention. Additionally, the transactivator fusion protein can include a third *oa polypeptide which promotes transport of the fusion protein to a cell nucleus. For *•o example, a nuclear localization signal having an amino acid sequence shown in SEQ ID No:5) can be incorporated into the fusion protein. The invention further *oo provides recombinant vectors and host cells comprising nucleic acid encoding a transactivator fusion protein of the invention. The invention still further provides transgenic and homologous recombinant organisms comprising nucleic acid encoding a transactivator fusion protein of the invention.

A transactivator of the invention is used to regulate the transcription of a target transcription unit composed of a nucleotide sequence to be transcribed operatively linked Q% to a minimal promoter sequence and at least one tet operator sequence. The nucleotide 4asequence to be transcribed may encode a protein of interest or an active RNA molecule antisense RNA molecule or ribozyme) and may be an exogenous or endogenous nucleotide sequence. A first nucleic acid encoding a transactivator of the invention and a second nucleic acid comprising a target transcription unit for the transactivator can be incorporated into a nucleic acid composition. The nucleic acid composition can be introduced into host cells or organisms transgenic and homologous recombinant animals and plants) to allow for tetracycline-inducible expression of the target nucleotide sequence to be transcribed.

In addition to providing a regulatory system for expression of a single nucleotide sequence to be transcribed, the invention also features novel target transcription units that enable coordinate or independent regulation of two or more nucleotide sequences to be transcribed. In one embodiment, coordinate regulation of two nucleotide sequences "to be transcribed is achieved using a transcription unit in which a first nucleotide sequence to be transcribed is operatively linked to the 5' end of a tet operator sequence(s) and a second nucleotide sequence to be transcribed is operatively linked to the 3' end of the same tet operator sequence(s) such that the first and second nucleotide sequences are transcribed in a divergent manner. Suitable bidirectional promoters for such bidirectional transcription units are shown in SEQ ID NOs: 6 and 7. Such transcription units are particularly useful for producing stoichiometric amounts of two subunits of a heterodimeric protein antibody chains) in the same cell or for coexpressing a gene of interest and a gene encoding a detectable marker in the same cell, thereby allowing for selection of cells expressing the gene of interest.

In another embodiment, independent regulation of two nucleotide sequences is achieved using a transcription unit in which a first nucleotide sequence to be transcribed is operatively linked to a tet operator of a first class type and a second nucleotide 4b sequence to be transcribed is operatively linked to a tet operator of a second, different class type. Two different transcriptional activator fusion proteins are then used to regulate the transcription of the two nucleotide sequences: one fusion protein binds to the first class of tet operator sequences in the presence of Tc, whereas the other fusion protein binds to the second class of tet operator sequences in the absence of Tc (or alternatively, the first fusion protein binds to the first class of tet operator sequences in the absence of Tc and the second fusion protein binds to the second class of tet operator sequences in the presence of Tc). This transcription unit is particularly useful for independently regulating expression of a therapeutic gene and a suicide gene in a host.

For example, expression of the therapeutic gene is stimulated in the presence of S•tetracycline and then when the therapy is completed, expression of the suicide gene is stimulated by removing the tetracycline.

~The invention further pertains to a method for stimulating transcription of a nucleotide sequence operatively linked to at least one tet operator sequence in a host cell 15 or animal which expresses a transactivator fusion protein of the invention. In a host cell, *o transcription is stimulated by contacting the cell with Tc or a Tc analogue. In a subject •*oo transgenic or homologous recombinant organisms), transcription is stimulated by administering Tc or a Tc analogue to the subject. Different Tc analogues, and variation 0***00 of the concentration of the inducing agent, can be used to modulate the level of induction of gene expression. Preferred Tc analogues for high level gene expression include anhydrotetracycline and doxycycline. The invention further provides a process for producing and isolating proteins of interest using the regulatory system of the invention is also provided.

The invention also pertains to tetracycline-regulated transcriptional inhibitors R 5 which are useful for inhibiting expression, in a highly controlled manner, of a gene linked to one or more tet operator sequences. The transcriptional inhibitors of the invention comprise a fusion protein composed of at least two polypeptides, a first polypeptide that binds to tet operator sequences and a heterologous second polypeptide that directly or indirectly inhibits transcription in eukaryotic cells. The heterologous the second polypeptide is derived from a different protein than the first polypeptide. Because the fusion proteins of the invention include a eukaryotic transcriptional silencer domain, they are anticipated to be more efficient at repressing transcription in eukaryotic cells than is a Tet Repressor alone.

In one embodiment of the invention, the first polypeptide of the inhibitor fusion protein binds to tet operator sequences in the absence but not the presence of tetracycline (Tc) or an analogue thereof the first polypeptide is preferably a Tet repressor, such as a Tnl 0-derived Tet repressor having an amino acid sequence shown in SEQ ID NO: 17). In the absence of tetracycline (or Tc analogue), this fusion protein binds to tet operator sequences operatively linked to a gene of interest, thereby inhibiting transcription of the gene of interest.

In another embodiment, the first polypeptide binds to tet operator sequences in the presence 15 but not the absence of tetracycline the first polypeptide is preferably a mutated Tet repressor, such as a Tnl 0-derived Tet repressor having an amino acid substitution at position 71, 95, 101 and/or 102). Preferably, the first polypeptide has an amino acid sequence shown S" in SEQ ID NO: 19. In the presence of tetracycline (or Tc analogue), this fusion protein binds to tet operator sequences operatively linked to a gene of interest, thereby inhibiting transcription of the gene of interest.

The second polypeptide can be a transcriptional "silencer" domain from a protein such as the v-erbA oncogene product, the Drosophila Krueppel protein, the retinoic acid receptor alpha, the thyroid hormone receptor alpha, the yeast Ssn6/Tupl protein complex, the Drosophila protein even-skipped, SIR1, NePI, the Drosophila dorsal protein, TSF3, SFI, the 25 Drosophila hunchback protein, the Drosophila knirps protein, WT1, Oct-2.1, the Drosophila engrailed protein, E4BP4 or ZF5. Preferred silencer domains include amino acid residues 403-466 of Krueppel (shown in SEQ ID NO: 21) and amino acid residues 364-635 ofv-erbA (shown in SEQ ID NO: 23).

The fusion proteins of the invention may further comprise additional polypeptides, such as a third polypeptide which promotes transport of the fusion protein into a cell nucleus a nuclear transport amino acid sequence).

This invention further provides isolated nucleic acid molecules encoding the transcriptional inhibitor fusion proteins of the invention and recombinant expression vectors containing these nucleic acid molecules in a form suitable for expression of the encoded transcriptional inhibitor fusion protein in a host cell. The invention still further provides host cells into which a recombinant expression vectors of the invention has been introduced.

Thus, a transcriptional inhibitor fusion protein is expressed in these host cells. The host cell can be, for example, a mammalian cell a human cell), a yeast cell, a fungal cell or an insect cell. Moreover, the host cell can be a fertilized non-human oocyte, in which case the -6host cell can be used to create a transgenic organism having cells that express the transcriptional inhibitor fusion protein. Still further, the recombinant expression vector can be designed to allow homologous recombination between the nucleic acid encoding the fusion protein and a target gene in a host cell. Such homologous recombination vectors can be used to create homologous recombinant animals that express a fusion protein of the invention.

In a preferred embodiment, the host cells (or cells of a host organism) also contain a nucleotide sequence to be transcribec operatively linked to at least one tet operator sequence a gene of interest whose expression can be regulated by Tc or an analogue thereof). To regulate transcription of the tet operator-linked gene of interest in these host cells, the concentration of Tc (or analogue thereof) in contact with the host cell is altered. For example, when the transcriptional inhibitor fusion protein binds to tet operator sequences in the absence of Tc, the concentration of Tc in contact with the cells is decreased to thereby inhibit transcription of the tet operator-linked gene of interest if cells are first cultured 15 in the presence of Tc, then Tc can be removed from the culture medium to inhibit transcription of the gene of interest). Alternatively, when the transcriptional inhibitor fusion protein binds to tet operator sequences in the presence of Tc, the concentration of Tc in *contact with the cells is increased to thereby inhibit transcription of the tet operator-linked gene of interest if cells are first cultured in the absence of Tc, then Tc can be added to the culture medium to inhibit transcription of the gene of interest).

SThe transcriptional inhibitor fusion proteins of the invention are useful for inhibiting gene expression in a variety of situations, as described further herein. In a particularly preferred embodiment, transcription of a tet operator(tetO)-linked gene of interest is regulated by a combination of tetracycline-regulated transcriptional inhibitor and activator fusion 25 proteins in the same host cell to allow for precise control of the expression level of the gene of interest. For example, an activator fusion protein that binds to tetO only in the presence of Tc and an inhibitor fusion protein that binds to tetO only in the absence of Tc are expressed in a host cell that contains a tetO-linked gene of interest. In the absence of Tc, basal levels of transcription of the gene of interest are inhibited by the inhibitor fusion protein. Upon contact of the cell with Tc (or analogue), transcription of the gene of interest is stimulated by the activator fusion protein. The activator and inhibitor fusion proteins of the invention can also be used in combination to regulate the expression of multiple tetO-linked genes of interest.

Novel kits for regulating the expression of a gene of interest are also within the scope of the invention. In one embodiment, the kits of the invention can include at least one nucleic acid molecule encoding a transactivator fusion protein of the invention and a target transcription unit into which a gene of interst can be cloned such that the gene is operatively linked to a tet operator sequence(s). Alternatively or additionally, the kits can include a nucleic acid molecule encoding a transcriptional inhibitor fusion protein of the invention.

-7- Target transcription units into which multiple genes of interest can be cloned, for coordinate or independent regulation, can also be included in kits of the invention.

Moreover, at least one tetracycline or tetracycline analogue may be included in the kits.

Accordingly, in one aspect of the present invention there is provided 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.

In another aspect of the present invention there is provided 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 analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.

In another aspect of the present invention there is provided a recombinant vector 15 comprising a nucleic acid molecule encoding a fusion protein of the invention in a form

S..

suitable for expression of the fusion protein in a host cell.

In still another aspect, the present invention provides a host cell comprising the .recombinant vector of the invention.

In yet another aspect of the present invention there is provided a method for stimulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the host cell of the invention comprising contacting the host cell with tetracycline or a tetracycline analogue.

In still yet another aspect of the present invention there is provided a non-human transgenic organism comprising a transgene comprising a nucleic acid molecule 7a encoding a fusion protein of the invention in a form suitable for expression of the fusion protein in cells of the non-human transgenic organism.

In another aspect of the present invention there is provided a method for stimulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the non-human transgenic organism of the invention, comprising administering tetracycline or a tetracycline analogue to the organism.

In another aspect of the present invention there is provided 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 a cells.

In anther aspect of the present invention there is provided a fusion protein which *ao inhibits transcription in eukaryotic cells, comprising a first polypeptide which binds to a tet operator sequence operatively linked to a heterologous second polypeptide which 15 inhibits transcription in eukaryotic cells.

In still another aspect of the present invention there is provided a method for S..modulating transcription of the nucleotide sequence operatively linked to the at least one a tet operator sequence in the host cell of the invention, comprising modulating the o concentration of tetracycline or a tetracycline analogue that is in contact with the host cell.

In still another aspect of the present invention there is provided a method for modulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the non-human transgenic organism of the invention, comprising modulating the concentration of tetracycline or a tetracycline analogue present in the S -T organism.

7b In another aspect of the present invention there is provided 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.

In another aspect of the present invention there is provided a non-human transgenic organism comprising: a first transgene encoding a first fusion protein which activates transcription, the *i 15 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 o: cells; .0 a second transgene encoding a second fusion protein which inhibits transcription, o 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 transgene comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

In still another aspect of the present invention there is provided a recombinant

S

c

R

l 25 vector for coordinate, bidirectional transcription of a first and a second nucleotide 7c sequence to be transcribed, the vector comprising a nucleotide sequence comprising in a 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.

10 In another aspect of the present invention there is provided a composition of matter .4 o 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, 15 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 1 transcribed, operatively linked to at least one tet operator sequence of a second class 4 4 type.

4 In still another aspect of the present invention there is provided 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 protein comprising a first polypeptide which S binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue 7doperatively 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.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Brief Description of the Drawings Figure 1 is a bar graph depicting the stimulation of luciferase activity in HR5-C11 .i 15 cells by tetracycline and different tetracycline analogues (l1 g/ml Cells were grown .in the absence or presence of the indicated tetracyclines for 3 days before luciferase activity was determined. Each solid and hatched bar represents the luciferase activity of S* a single culture dish.

Figure 2 is a graph depicting the relative luciferase activity in HR5-C11 cells when incubated with different concentrations of doxycycline. The results of three independent experiments are shown.

Figure 3 is a graph depicting the kinetics of induction of luciferase activity in HR5-C11 cells by doxycycline. HR5-C11 cultures were exposed to 1lpg/ml of doxycycline and luciferase activity was measured after different time intervals; S~ \25 cultures containing doxycycline, cultures grown in the absence of antibiotic.

7e- Figure 4 shows the amino acid sequences of various classes of Tet repressors, illustrating the homology between the amino acid sequences of different classes of Tet repressors, as compared to class B Tet repressors Tnl0-derived). Amino acid positions in other classes of Tet repressors that are identical to class B are indicated by a dash.

Figure 5 shows the nucleotide sequences of tet operators of different classes: class A (SEQ ID NO:11), class B (SEQ ID NO: 12), class C (SEQ ID NO: 13), class D (SEQ ID NO: 14) and class E (SEQ ID NO: Figure 6 is a schematic diagram of a bidirectional promoter construct for coordinate regulation of two genes of interest operatively linked to the same tet operators for regulation by a tetracycline-regulated transcriptional activator.

Figure 7A (SEQ ID NO:6) shows the nucleotide sequence of a bidirectional promoter region for coordinate regulation of two genes of interest by a tetracyclineo ~regulated transcriptional activator.

Figure 7B (SEQ ID NO:7) shows the nucleotide sequence of a bidirectional *i promoter region for coordinate regulation of two genes of interest by a tetracyclineregulated transcriptional activator.

1: Figure 8 is two graphs depicting coordinate expression of luciferase and galactosidase activity by a tetracycline-regulated transcriptional activator.

Figure 9A-B are schematic diagrams of self-regulating promoters for expression of tetracycline-regulated transcriptional activators (tTA). Panel A illustrates self-regulation of expression of a wild-type Tet repressor-containing transactivator fusion protein that binds to tet operators in the absence of Tc. Panel B illustrates self-regulation of expression of a mutated Tet repressor-containing transactivator fusion protein that binds to tet operators in the presence of Tc.

Figure 10 is a schematic diagram of the negative and positive regulation of a tet operator (tetOwt)-linked gene of interest by a tetracycline-regulated transcriptional inhibitor protein (tSD) and a tetracycline-inducible transcriptional activator fusion protein (rtTA), respectively, in the presence of increasing concentrations of the tetracycline analogue doxycycline.

Figure 11 is a schematic diagram of the construction of TetR-silencer domain fusion contructs by in-frame fusion of nucleic acid encoding either a Krueppel or v-erbA silencer domain to the 3' end of nucleic acid encoding a Tet repressor (tetR gene).

Figure 12 is a graphic representation of the expression of luciferase activity in mice transgenic for the luciferase reporter gene alone (checked columns at right) or double transgenic animals carrying the luciferase reporter gene and a tTAR transgene, either in the :*absence of doxycycline (dark columns in middle) or in the presence of doxycycline (light 15 columns at left).

Detailed Description of the Invention *This invention pertains to nucleic acid molecules and proteins which can be used to regulate the expression of genes in eukaryotic cells or animals in a highly controlled manner.

20 Regulation of gene expression by the system of the invention involves at least two components: A gene which is operatively linked to a regulatory sequence and a protein which, in either the presence or absence of an inducible agent, binds to the regulatory sequence and either activates or inhibits transcription of the gene. The system of the invention utilizes components of the Tet repressor/operator/inducer system of prokaryotes to stimulate gene expression in eukaryotic cells.

Various aspects of the invention pertain to fusion proteins which are capable of either activating or inhibiting gene transcription when bound to tet operator (tetO) sequences, but which bind to tet operator sequences only in the presence or, alternatively, in the absence of tetracycline, or an analogue thereof. Thus, in a host cell, transcription of a gene operatively linked to a tet operator sequence(s) is stimulated or inhibited by a fusion protein of the invention by altering the concentration of tetracycline (or analogue) in contact with the host cell adding or removing tetracycline from a culture medium, or administering or ceasing to administer tetracycline to a host organism, etc.).

The invention further pertains to target transcription units for regulation by the fusion protein of the invention. In addition to allowing for regulation of a single tet-operator linked gene of interest, the invention also provides novel transcription units containing two or more genes to be transcribed that can be regulated in either a coordinate or independent manner by a transactivator fusion protein of the invention. Methods for stimulating or inhibiting transcription of a gene using tetracycline (or analogues thereof), and kits which contain the -9components of the regulatory system described herein, are also encompassed by the invention.

In the following subsections, the nucleic acids and proteins comprising the components of the inducible regulatory system of the invention, and their interrelationship, are discussed in greater detail. The subsections are as follows: I. Tetracycline-Inducible Transcriptional Activators A. The first polypeptide of the transactivator fusion protein B. The second polypeptide of the transactivator fusion protein C. A third polypeptide of the transactivator fusion protein II. Expression of a Transactivator Fusion Protein A. Expression vectors B. Host cells 15 C. Introduction of nucleic acid into host cells D. Transgenic Organisms E. Homologous Recombinant Organisms III. Target Transcription Units Regulated by a Tetracycline-Inducible Transactivator 20 A. Regulation of expression of tet operator-linked nucleotide sequences B. Coordinate regulation of two nucleotide sequences C. Independent regulation of mulitple nucleotide sequences D. Combined coordinate and independent regulation of multiple nucleotide sequences IV. Tetracycline-Regulated Transcriptional Inhibitors A. The first polypeptide of the transcritional inhibitor fusion protein B. The second polypeptide of the-transcritional inhibitor fusion protein C. A third polypeptide of the transcritional inhibitor fusion protein D. Expression of the transcriptional inhibitor fusion protein V. Kits of the Invention VI. Regulation of Gene Expression by Tetracycline or Analogues Thereof A. Stimulation of gene expression by transactivator fusion proteins B. Inhibition of gene expression by transcriptional inhibitor fusion proteins C. Combined positive and negative regulation of gene expression VII. Applications of the Invention A. Gene Therapy B. Production of Proteins in Vitro C. Production of Proteins in Vivo D. Animal Models of Human Disease E. Production of Stable Cell Lines for Cloning I. Tetracvcline-Inducible Transcriptional Activators In the inducible regulatory system of the invention, transcription of a gene is activated by a transcriptional activator protein, also referred to herein simply as a transactivator. The transactivator of the invention is a fusion protein. One aspect of the invention thus pertains to fusion proteins and nucleic acids DNA) encoding fusion proteins. The term "fusion protein" is intended to describe at least two polypeptides, typically from different sources, which are operatively linked. With regard to the polypeptides, the term "operatively linked" 15 is intended to mean that the two polypeptides are connected in manner such that each polypeptide can serve its intended function. Typically, the two polypeptides are covalently attached through peptide bonds. The fusion protein is preferably produced by standard recombinant DNA techniques. For example, a DNA molecule encoding the first polypeptide is ligated to another DNA molecule encoding the second polypeptide, and the resultant hybrid 20 DNA molecule is expressed in a host cell to produce the fusion protein. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered the DNA molecules are ligated to each .other in-frame).

"25 A. The first polypeptide of the transactivator fusion protein The transactivator fusion protein of the invention is composed, in part, of a first polypeptide which binds to a tet operator sequence in the presence of tetracycline or an analogue thereof. The first polypeptide of the fusion protein is preferably a mutated Tet repressor. The term "mutated Tet repressor" is intended to include polypeptides having an amino acid sequence which is similar to a wild-type Tet repressor but which has at least one amino acid difference from the wild-type Tet repressor. The term "wild-type Tet repressor" is intended to describe a protein occurring in nature which represses transcription from tet operator sequences in prokaryotic cells in the absence of Tc. The amino acid difference(s) between a mutated Tet repressor and a wild-type Tet repressor may be substitution of one or more amino acids, deletion of one or more amino acids or addition of one or more amino acids. The mutated Tet repressor of the invention has the following functional properties: 1) the polypeptide can bind to a tet operator sequence, it retains the DNA binding specificity of a wild-type Tet repressor, and 2) it is regulated in a reverse manner by -11 tetracycline than a wild-type Tet repressor, Le., the mutated Tet repressor binds to a ter operator sequence only the presence of Tc (or Tc analogue) rather than in the absence of Tc.

In a preferred embodiment, a mutated Tet repressor having the functional properties described above is created by substitution of amino acid residues in the sequence of a wildtype Tet repressor. For example, as described in Example 1, a Tnl0-derived Tet repressor having amino acid substitutions at amino acid positions 71, 95, 101 and 102 has the desired functional properties and thus can be used as the first polypeptide in the transactivator fusion protein of the invention. The amino acid sequence of this mutated Tet repressor is shown in SEQ ID NO: 2 (positions 1-207). In one embodiment of the mutated Tet repressor, position 71 is mutated from glutamic acid to lysine, position 95 is mutated from aspartic acid to asparagine, position 101 is mutated from leucine to serine and position 102 is mutated from glycine to aspartic acid, although the invention is not limited to these particular mutations.

Mutation of fewer than all four of these amino acid positions may be sufficient to acheive a Tet repressor with the desired functional properties. Accordingly, a Tet repressor is 15 preferably mutated at at least one of these positions. Other amino acid substitutions, deletions or additions at these or other amino acid positions which retain the desired i* functional properties of the mutated Tet repressor are within the scope of the invention. The crystal structure of a Tet repressor-tetracycline complex, as described in Hinrichs, W. et al.

(1994) Science 264:418-420, can be used for rational design of mutated Tet repressors.

20 Based upon this structure, amino acid position 71 is located outside the tetracyline binding o pocket, suggesting mutation at this site may not be necessary to achieve the desired functional properties of a mutated Tet repressor of the invention. In contrast, amino acid positions95, 101 and 102 are located within the conserved tetracyline binding pocket. Thus, the tetracycline binding pocket of a Tet repressor may be targeted for mutation to create a mutated Tet repressor of the invention.

Additional mutated Tet repressors for incorporation into a fusion protein of the invention can be created according to the teachings of the invention. A number of different classes of Tet repressors have been described, A, B, C, D and E (of which theTnl0encoded repressor is a class B repressor). The amino acid sequences of the different classes of Tet repressors share a high degree of homology 40-60 across the length of the proteins), including in the region encompassing the above-described mutations. The amino acid sequences of various classes of Tet repressors are shown and compared in Figure 4, and are also described in Tovar, K. et al. (1988) Mol. Gen. Genet. 21:76-80. Accordingly, equivalent mutations to those described above for the TnlO-derived Tet repressor can be made in other classes of Tet repressors for inclusion in a fusion protein of the invention. For example, amino acid position 95, which is an aspartic acid in all five repressor classes, can be mutated to asparagine in any class of repressor. Similarly, position 102, which is glycine in all five repressor classes, can be mutated to aspartic acid in any class of repressor.

Additional suitable equivalent mutations will be apparent to those skilled in the art and can be -12created and tested for functionality by procedures described herein. Nucleotide and amino acid sequences of Tet repressors of the A, C, D and E classes are disclosed in Waters, SH. et al. (1983) Nucl. Acids Res 11:6089-6105, Unger, B. et al. (1984) Gene 31: 103-108, Unger, B. et al. (1984) Nucl Acids Res. 12:7693-7703 and Tovar, K. et al. (1988) Mol. Gen. Genet.

215:76-80, respectively. These wild-type sequences can be mutated according to the teachings of the invention for use in the inducible regulatory system described herein.

Alternative to the above-described mutations, additional suitable mutated Tet repressors having the desired functional properties described above) can be created by mutagenesis of a wild type Tet repressor and selection as described in Example 1. The nucleotide and amino acid sequences of wild-type class B Tet repressors are disclosed in Hillen, W. and Schollmeier, K. (1983) Nucl. Acids Res. 11:525-539 and Postle, K. et al.

(1984) Nucl. Acids Res. 12:4849-4863. The nucleotide and amino acid sequences of wildtype class A, C, D and E type repressors are cited above. A mutated Tet repressor can be created and selected, for example as follows: a nucleic acid DNA) encoding a wild-type 15 Tet repressor is subjected to random mutagenesis and the resultant mutated nucleic acids are incorporated into an expression vector and introduced into a host cell for screening. A screening assay is used which allows for selection of a Tet repressor which binds to a tet operator sequence only in the presence of tetracycline. For example, a library of mutated nucleic acids in an expression vector can be introduced into an E. coli strain in which tet 20 operator sequences control the expression of a gene encoding a Lac repressor and the Lac repressor controls the expression of a gene encoding an selectable marker drug resistance). Binding of a Tet repressor to tet operator sequences in the bacteria will inhibit expression of the Lac repressor, thereby inducing expression of the selectable marker gene.

Cells expressing the marker gene are selected based upon the selectable phenotype drug resistance). For wild-type Tet repressors, expression of the selectable marker gene will occur in the absence ofTc. A nucleic acid encoding a mutated Tet repressor is selected using this system based upon the ability of the nucleic acid to induce expression of the selectable marker gene in the bacteria only in the presence of Tc.

A first polypeptide of the transactivator fusion protein the mutated Tet repressor) has the property of binding specifically to a tet operator sequence. Each class of Tet repressor has a corresponding target tet operator sequence. Accordingly, the term "tet operator sequence" is intended to encompass all classes of tet operator sequences, e.g. class A, B, C, D, and E. Nucleotide sequences of these five classes of tet operators are shown in Figure 5 and SEQ ID NOs: 11-15, and are described in Waters, S.H. et al. (1983) cited supra, Hillen, W. and Schollenmeier, K. (1983) cited supra, StOber, D. and Bujard, H. (1981) Proc.

Natl. Acad Sci. USA 71:167-171, Unger, B. et al. (1984) cited supra and Tovar, K. et al.

(1988) cited supra. In a preferred embodiment, the mutated Tet repressor is a Tnl0-encoded repressor class B) and the tet operator sequence is a class B tet operator sequence.

-13- Alternatively, a mutated class A Tet repressor can be used with a class A tet operator sequence, and so on for the other classes of Tet repressr/operators.

Another approach for creating a mutated Tet repressor which binds to a class A tet operator is to further mutate the already mutated Tnl -derived Tet repressor described herein (a class B repressor) such that it no longer binds efficiently to a class B type operator but instead binds efficiently to a class A type operator. It has been found that nucleotide position 6 of class A or B type operators is the critical nucleotide for recognition of the operator by its complimentary repressor (position 6 is a G/C pair in class B operators and an A/T pair in class A operators) (see Wissman et al. (1988) J. Mol. Biol. 202:397-406). It has also been found that amino acid position 40 of a class A or class B Tet repressor is the critical amino acid residue for recognition of position 6 of the operator (amino acid position 40 is a threonine in class B repressors but is an alanine in class A repressors). It still further has been found that substitution of Thr40 of a class B repressor with Ala alters its binding specificity such that the repressor can now bind a class A operator (similarly, substitution of 15 Ala40 of a class A repressor with Thr alters its binding specificity such that the repressor can now bind a class B operator) (see Altschmied et al. (1988) EMBO J. 2:4011-4017).

Accordingly, one can alter the binding specificity of the mutated Tnl -derived Tet repressor "disclosed herein by additionally changing amino acid residue 40 from Thr to Ala by standard molecular biology techniques site directed mutagenesis).

20 A mutated Tet repressor having specific mutations at positions 71, 95, 101 and/or 102, as described above) can be created by introducing nucleotide changes into a nucleic acid encoding a wild-type repressor by standard molecular biology techniques, e.g.

site directed mutagenesis or PCR-mediated mutagenesis using oligonucleotide primers incorporating the nucleotide mutations. Alternatively, when a mutated Tet repressor is identified by selection from a library, the mutated nucleic acid can be recovered from the library vector. To create a transactivator fusion protein of the invention, a nucleic acid encoding a mutated Tet repressor is then ligated in-frame to another nucleic acid encoding a transcriptional activation domain and the-fusion construct is incorporated into a recombinant expression vector. The transactivator fusion protein can be expressed by introducing the recombinant expression vector into a host cell or animal.

B. The second polypeptide of the transactivator fusion protein The first polypeptide of the transactivator fusion protein is operatively linked to a second polypeptide which directly or indirectly activates transcription in eukaryotic cells. To operatively link the first and second polypeptides, typically nucleotide sequences encoding the first and second polypeptides are ligated to each other in-frame to create a chimeric gene encoding a fusion protein, although the first and second polypeptides can be operatively linked by other means that preserve the function of each polypeptide chemically crosslinked). In a preferred embodiment, the second polypeptide of the transactivator itself -14possesses transcriptional activation activity the second polypeptide directly activates transcription). In another embodiment, the second polypeptide activates transcription by an indirect mechanims, through recruitment of a transcriptional activation protein to interact with the fusion protein. Accordingly, the term "a polypeptide which activates transcription in eukaryotic cells" as used herein is intended to include polypeptides which either directly or indirectly activates transcription.

Polypeptides which can function to activate transcription in eukaryotic cells are well known in the art. In particular, transcriptional activation domains of many DNA binding proteins have been described and have been shown to retain their activation function when the domain is transferred to a heterologous protein. A preferred polypeptide for use in the fusion protein of the invention is the herpes simplex virus virion protein 16 (referred to herein as VP16, the amino acid sequence of which is disclosed in Triezenberg, S.J. et al. (1988) Genes Dev. 2:718-729). In one embodiment, about 127 of the C-terminal amino acids of VP16 are used. For example, a polypeptide having an amino acid sequence shown in SEQ ID 15 NO: 2 (positions 208-335) can be used as the second polypeptide in the fusion protein. In another embodiment, at least one copy of about 11 amino acids from the C-terminal region of VP 16 which retain transcriptional activation ability is used as the second polypeptide.

Preferably, a dimer of this region about 22 amino acids) is used. Suitable C-terminal peptide portions of VP16 are described in Seipel, K. et al. (EMBO J. (1992) 13:4961-4968).

20 For example, a dimer of a peptide having an amino acid sequence shown in SEQ ID NO: 4 S* (encoded by a nucleotide sequence shown in SEQ ID NO: 3) can be used as the second polypeptide in the fusion protein.

Other polypeptides with transcriptional activation ability in eukaryotic cells can be used in the fusion protein of the invention. Transcriptional activation domains found within 25 various proteins have been grouped into categories based upon similar structural features.

Types of transcriptional activation domains include acidic transcription activation domains, proline-rich transcription activation domains, serine/threonine-rich transcription activation domains and glutamine-rich transcription.activation domains. Examples of acidic transcriptional activation domains include the VPI 6 regions already described and amino acid residues 753-881 of GAL4. Examples of proline-rich activation domains include amino acid residues 399-499 of CTF/NFI and amino acid residues 31-76 of AP2. Examples of serine/threonine-rich transcription activation domains include amino acid residues 1-427 of ITFI and amino acid residues 2-451 of ITF2. Examples of glutamine-rich activation domains include amino acid residues 175-269 of Octl and amino acid residues 132-243 of Spl. The amino acid sequences of each of the above described regions, and of other useful transcriptional activation domains, are disclosed in Seipel, K. et al. (EMBO J. (1992) .:14961-4968).

In addition to previously described transcriptional activation domains, novel transcriptional activation domains, which can be identified by standard techniques, are within the scope of the invention. The transcriptional activation ability of a polypeptide can be assayed by linking the polypeptide to another polypeptide having DNA binding activity and determining the amount of transcription of a target sequence that is stimulated by the fusion protein. For example, a standard assay used in the art utilizes a fusion protein of a putative transcriptional activation domain and a GAL4 DNA binding domain amino acid residues 1-93). This fusion protein is then used to stimulate expression of a reporter gene linked to GAL4 binding sites (see Seipel, K. et al. (1992) EMBO J. 11:4961-4968 and references cited therein).

In another embodiment, the second polypeptide of the fusion protein indirectly activates transcription by recruiting a transcriptional activator to interact with the fusion protein. For example, a mutated tetR of the invention can be fused to a polypeptide domain a dimerization domain) capable of mediating a protein-protein interaction with a transcriptional activator protein, such as an endogenous activator present in a host cell. It has been demonstrated that functional associations between DNA binding domains and 15 transactivation domains need not be covalent (see Fields and Song (1989) Nature 3A4:245-247; Chien et al. (1991) Proc. Natl. Acad Sci. USA 88:9578-9582; Gyuris et al.

(1993) Cell 5:791-803; and Zervos, A.S. (1993) Cell 22:223-232). Accordingly, the second polypeptide of the fusion protein may not directly activate transcription but rather may form a stable interaction with an endogenous polypeptide bearing a compatible protein-protein 20 interaction domain and transactivation domain. Examples of suitable interaction (or dimerization) domains include leucine zippers (Landschulz et al. (1989) Science 24: 1681- 1688), helix-loop-helix domains (Murre, C. et al. (1989) Cell 8:537-544) and zinc finger domains (Frankel, A.D. et al. (1988) Science 24Q:70-73). Interaction of a dimerization domain present in the fusion protein with an endogeneous nuclear factor results in recruitment of the transactivation domain of the nuclear factor to the fusion protein, and thereby to a tet operator sequence to which the fusion protein is bound.

C. A third polypeptide of the transactivator fusion protein In addition to a mutated Tet repressor and a transcriptional activation domain, a fusion protein of the invention can contain an operatively linked third polypeptide which promotes transport of the fusion protein to a cell nucleus. Amino acid sequences which, when included in a protein, function to promote transport of the protein to the nucleus are known in the art and are termed nuclear localization signals (NLS). Nuclear localization signals typically are composed of a stretch of basic amino acids. When attached to a heterologous protein a fusion protein of the invention), the nuclear localization signal promotes transport of the protein to a cell nucleus. The nuclear localization signal is attached to a heterologous protein such that it is exposed on the protein surface and does not interfere with the function of the protein. Preferably, the NLS is attached to one end of the protein, e.g. the N-terminus. The amino acid sequence of a non-limiting exampleof an NLS that can be included in a fusion -16protein of the invention is shown in SEQ ID NO: 5. Preferably, a nucleic acid encoding the nuclear localization signal is spliced by standard recombinant DNA techniques in-frame to the nucleic acid encoding the fusion protein at the 5' end).

The plasmid pUHD17-1 (described in further detail in Example which comprises a transactivator of the invention having the nucleotide sequence shown in SEQ ID NO: 1, has been deposited on July 8, 1994 under the provisions of the Budapest Treaty at the Deutsche Sammlung Von Mikroorganismen und ZellKulturen GmbH (DSM) in Braunschweig, Germany and assigned deposit number DSM 9279.

11. Expression of a Transactivator Fusion Protein A. Expression Vectors A nucleic acid of the invention encoding a transactivator fusion protein, as described 15 above, can be incorporated into a recombinant expression vector in a form suitable for expression of the fusion protein in a host cell. The term "in a form suitable for expression of the fusion protein in a host cell" is intended to mean that the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid encoding the fusion protein in a manner which allows for transcription of the nucleic acid into mRNA and 20 translation of the mRNA into the fusion protein. The term "regulatory sequence" is artrecognized and intended to include promoters, enhancers and other expression control elements polyadenylation signals). Such regulatory sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the amount of fusion protein to be expressed.

When used in mammalian cells, a recombinant expression vector's control functions are often provided by viral genetic material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Use of viral regulatory elements to direct expression of the fusion protein can allow for high level constitutive expression of the fusion protein in a variety of host cells. In a preferred recombinant expression vector, the sequences encoding the fusion protein are flanked upstream by the human cytomegalovirus IE promoter and downstream by an poly(A) signal. For example, an expression vector similar to that described in Example 1 can be used. The human cytomegalovirus IE promoter is described in Boshart et al. (1985) Cell 41:521-530. Other ubiquitously expressing promoters which can be used include the HSV-Tk promoter (disclosed in McKnight et al. (1984) Cell 32:253-262) and j-actin promoters the human p-actin promoter as described by Ng et al. (1985) Mol. Cell. Biol.

5:2720-2732).

-17- Alternatively, the regulatory sequences of the recombinant expression vector can direct expression of the fusion protein preferentially in a particular cell type, tissuespecific regulatory elements can be used. Non-limiting examples of tissue-specific promoters which can be used include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.

413235-275), in particular promoters ofT cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 1:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters the neurofilament promoter, Byme and Ruddle (1989) Proc. Natl. Acad Sci. USA 86:5473-5477), pancreasspecific promoters (Edlund et al. (1985) Science 2M0:912-916), and mammary gland-specific promoters milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the S. a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

15 Alternatively, a self-regulating construct encoding a transactivator fusion protein can be created. To accomplish this, nucleic acid encoding the fusion protein is operatively linked to a minimal promoter sequence and at least one tet operator sequence. For example, the nucleic acid of SEQ ID NO: 1 can be linked to a promoter having a nucleotide sequence shown in SEQ ID NO: 8, 9 or 10 (the nucleic acids of SEQ ID NOs: 8 and 9 comprise a 20 minimal CMV promoter and ten tet operators; the nucleic acids of SEQ ID NO: 10 comprises a TK promoter and ten tet operators). A schematic diagram of such a self-regulating construct is shown in Figure 9B. When this nucleic acid is introduced into a cell in a recombinant expression vector), a small amount of basal transcription of the transactivator gene is likely to occur due to "leakiness". In the presence ofTc (or analogue thereof) this 25 small amount of the transactivator fusion protein will bind to the tet operator sequence(s) upstream of the nucleotide sequence encoding the transactivator and stimulate additional transcription of the nucleotide sequence encoding the transactivator, thereby leading to further production of the transactivator fusion protein in the cell. It will be appreciated by those skilled in the art that such a self-regulating promoter can also be used in conjunction with other tetracycline-regulated transactivators, such as the wild-type Tet repressor fusion protein (tTA) described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 81:5547-5551, which binds to tet operators in the absence of Tc (as illustrated in Figure 9A).

When used in conjunction with this transactivator, self-regulated transcription of the nucleotide sequence encoding this transactivator is stimulated in the absence of Tc. The plasmid pUHD15-3, which comprises nucleotide sequences encoding the tTA described in Gossen and Bujard (1992), cited supra, operatively linked to a self-regulating promoter, has been deposited on July 8, 1994 under the provisions of the Budapest Treaty at the Deutsche Sammlung Von Mikroorganismen und ZellKulturen GmbH (DSM) in Braunschweig, Germany and assigned deposit number DSM 9280.

-18- In one embodiment, the recombinant expression vector of the invention is a plasmid, such as that described in Example 1. Alternatively, a recombinant expression vector of the invention can be a virus, or portion thereof, which allows for expression of a nucleic acid introduced into the viral nucleic acid. For example, replication defective retroviruses, adenoviruses and adeno-associated viruses can be used. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include /Crip, Cre, \y 2 and yAm.

The genome of adenovirus can be manipulated such that it encodes and expresses a transactivator fusion protein but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable 15 adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of *adenovirus Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.

Alternatively, an adeno-associated virus vector such as that described in Tratschin et al.

(1985) Mol. Cell. Biol. 5:3251-3260 can be used to express a transactivator fusion protein.

20 B. Host Cells A fusion protein of the invention is expressed in a eukaryotic cell by introducing nucleic acid encoding the fusion protein into a host cell, wherein the nucleic acid is in a form suitable for expression of the fusion protein in the host cell. For example, a recombinant expression vector of the invention, encoding the fusion protein, is introduced into a host cell.

Alternatively, nucleic acid encoding the fusion protein which is operatively linked to regulatory sequences promoter sequences) but without additional vector sequences can be introduced into a host cell. As used herein, the term "host cell" is intended to include any eukaryotic cell or cell line so long as the cell or cell line is not incompatible with the protein to be expressed, the selection system chosen or the fermentation system employed. Nonlimiting examples of mammalian cell lines which can be used include CHO dhfr- cells (Urlaub and Chasin (1980) Proc. Natl. Acad Sci. USA 22:4216-4220), 293 cells (Graham et al. (1977) J. Gen Virol. 36: pp59) or myeloma cells like SP2 orNSO (Galfre and Milstein (1981) Meth. Enzymol. 23(B):3-46).

In addition to cell lines, the invention is applicable to normal cells, such as cells to be modified for gene therapy purposes or embryonic cells modified to create a transgenic or homologous recombinant animal. Examples of cell types of particular interest for gene therapy purposes include hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, neuronal cells and skin epithelium and airway epithelium. Additionally, for transgenic or homologous recombinant animals, embryonic stem cells and fertilized oocytes can be -19modified to contain nucleic acid encoding a transactivator fusion protein. Moreover, plant cells can be modified to create transgenic plants.

The invention is broadly applicable and encompasses non-mammalian eukaryotic cells as well, including insect Sp. frugiperda), yeast S. cerevisiae, S. pombe, P.

pastoris, K. lactis, H. polymorpha; as generally reviewed by Fleer, R. (1992) Current Opinion in Biotechnology 3{5:486-496)), fungal and plant cells. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et a, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz t L, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). The fusion protein can be expressed in insect cells using baculovirus expression vectors as described in O'Reilly et al. (1992) Baculovirus Expression Vectors: A Laboratory Manual, Stockton Press).

Baculovirus vectors available for expression of proteins in cultured insect cells SF 9 cells) include the pAc series (Smith at aL (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, and Summers, (1989) Virology 17Q:31-39).

C. Introduction ofNucleic Acid into a Host Cell Nucleic acid encoding the fusion protein can be introduced into a host cell by standard techniques for transfecting eukaryotic cells. The term "transfecting" or "transfection" is intended to encompass all conventional techniques for introducing nucleic acid into host 20 cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation and microinjection. Suitable methods for transfecting host cells can be found in Sambrook et aL (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

The number of host cells transformed with a nucleic acid of the invention will depend, 25 at least in part, upon the type of recombinant expression vector used and the type of transfection technique used. Nucleic acid can be introduced into a host cell transiently, or more typically, for long term regulation of gene expression, the nucleic acid is stably integrated into the genome of the host cell or remains as a stable episome in the host cell.

Plasmid vectors introduced into mammalian cells are typically integrated into host cell DNA at only a low frequency. In order to identify these integrants, a gene that contains a selectable marker drug resistance) is generally introduced into the host cells along with the nucleic acid of interest. Preferred selectable markers include those which confer resistance to certain drugs, such as G418 and hygromycin. Selectable markers can be introduced on a separate plasmid from the nucleic acid of interest or, are introduced on the same plasmid. Host cells transfected with a nucleic acid of the invention a recombinant expression vector) and a gene for a selectable marker can be identified by selecting for cells using the selectable marker. For example, if the selectable marker encodes a gene conferring neomycin resistance, host cells which have taken up nucleic acid can be selected with G418. Cells.that have incorporated the selectable marker gene will survive, while the other cells die.

A host cell transfected with a nucleic acid encoding a fusion protein of the invention can be further transfected with one or more nucleic acids which serve as the target for the fusion protein. The target nucleic acid comprises a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence (described in more detail in Section III below).

Nucleic acid encoding the fusion protein of the invention can be introduced into eukaryotic cells growing in culture in vitro by conventional transfection techniques calcium phosphate precipitation, DEAE-dextran transfection, electroporationetc.). Nucleic acid can also be transferred into cells in vivo, for example by application of a delivery mechanism suitable for introduction of nucleic acid into cells in vivo, such as retroviral vectors (see Ferry, N et al. (1991) Proc. Natl. Acad Sci. USA 8818377-8381; and Kay, MA. et al. (1992) Human Gene Therapy 3:641-647), adenoviral vectors (see Rosenfeld, M.A. (1992) Celli68;143-155; and Herz, J. and Gerard, R.D. (1993) Proc. Natl. Acad Sci.

USA 9Q02812-2816), receptor-mediated DNA uptake (see Wu, G. and Wu, C.H. (1988) 15 J. Biol. Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 26L2963-967; and U.S. Patent No. 5,166,320), direct injection of DNA (see Acsadi et al. (1991) Nature 332- 815-818; and Wolff et al. (1990) Science 2421465-1468) or particle bombardment (see Cheng, L.

et al. (1993) Proc. Natl. Acad Sci. USA 90:4455-4459; and Zelenin, A.V. et al. (1993) FEBS \Letters 315:29-32). Thus, for gene therapy purposes, cells can be modified in vitro and 20 administered to a subject or, alternatively, cells can be directly modified in vivo.

D. Transgenic Organisms Nucleic acid a transactivator fusion protein can transferred into a fertilized oocyte of a non-human animal to create a transgenic animal which expresses the fusion protein of the 25 invention in one or more cell types. A transgenic animal is an animal having cells that contain a transgene, wherein the transgene was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic, stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. In one embodiment, the nonhuman animal is a mouse, although the invention is not limited thereto. In other embodiments, the transgenic animal is a goat, sheep, pig, cow or other domestic farm animal.

Such transgenic animals are useful for large scale production of proteins (so called "gene pharming").

A transgenic animal can be created, for example, by introducing a nucleic acid encoding the fusion protein (typically linked to appropriate regulatory elements, such as a constitutive or tissue-specific enhancer) into the male pronuclei of a fertilized oocyte, by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to -21increase the efficiency of expression of the transgene. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and Hogan, B. et a., (1986) A Laboratory Manual, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory. A transgenic founder animal can be used to breed additional animals carrying the transgene. Transgenic animals carrying a transgene encoding the fusion protein of the invention can further be bred to other transgenic animals carrying other transgenes, to a transgenic animal which contains a gene operatively linked to a tet operator sequence (discussed in more detail in Section III below).

It will be appreciated that, in addition to transgenic animals, the regulatory system described herein can be applied to other transgenic organisms, such as transgenic plants.

Transgenic plants can be made by conventional techniques known in the art. Accordingly, the invention encompasses non-human transgenic organisms, including animals and plants, that contains cells which express the transactivator fusion protein of the invention a nucleic acid encoding the transactivator is incorporated into one or more chromosomes in cells of the transgenic organism).

E. Homologous Recombinant Organisms The invention also provides a homologous recombinant non-human organism 20 expressing the fusion protein of the invention. The term "homologous recombinant organism" as used herein is intended to describe an organism, e.g. animal or plant, containing a gene which has been modified by homologous recombination between the gene and a DNA molecule introduced into a cell of the animal, an embryonic cell of the animal. In one embodiment, the non-human animal is a mouse, although the invention is not limited thereto.

25 An animal can be created in which nucleic acid encoding the fusion protein has been introduced into a specific site of the genome, the nucleic acid has homologously recombined with an endogenous gene.

To create such a homologous recombinant animal, a vector is prepared which contains DNA encoding the fusion protein flanked at its 5' and 3' ends by additional nucleic acid of a eukaryotic gene at which homologous recombination is to occur. The additional nucleic acid flanking that encoding the fusion protein is of sufficient length for successful homologous recombination with the eukaryotic gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see Thomas, K.R. and Capecchi, M. R.

(1987) Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal a mouse) to form aggregation chimeras (see Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed.

-22- (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harbouring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA. These "germline transmission" animals can further be mated to animals carrying a gene operatively linked to at least one tet operator sequence (discussed in more detail in Section I below).

In addition to the homologous recombination approaches described above, enzymeassisted site-specific integration systems are known in the art and can be applied to the components of the regulatory system of the invention to integrate a DNA molecule at a predetermined location in a second target DNA molecule. Examples of such enzyme-assisted integration systems include the Cre recombinase-lox target system as described in Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 12:7905-7909) and the FLP recombinase-FRT target system as described in Dang, D.T. and Perrimon, N. (1992) Dev. Genet. 11:367- 15 375; and Fiering, S. et al. (1993) Proc. Natl. Acad Sci. USA 90:8469-8473).

III. Target Transcription Units Regulated by a Tetracycline-Inducible Transactivator A fusion protein of the invention is used to regulate the transcription of a target nucleotide sequence. This target nucleotide sequence is operatively linked to a regulatory 20 sequence to which the fusion protein binds. More specifically, the fusion protein regulates expression of a nucleotide sequence operatively linked to at least one tet operator sequence.

Accordingly, another aspect of the invention relates to target nucleic acids DNA molecules) comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence. Such nucleic acids are also referred to herein as tet-regulated 25 transcription units (or simply transcription units).

Within a transcription unit, the "nucleotide sequence to be transcribed" typically includes a minimal promoter sequence which is not itself transcribed but which serves (at least in part) to position the transcriptional machinery for transcription. The minimal promoter sequence is linked to the transcribed sequence in a 5' to 3' direction by phosphodiester bonds the promoter is located upstream of the transcribed sequence) to form a contiguous nucleotide sequence. Accordingly, as used herein, the terms "nucleotide sequence to be transcribed" or "target nucleotide sequence" are intended to include both the nucleotide sequence which is transcribed into mRNA and an operatively linked upstream minimal promoter sequence. The term "minimal promoter" is intended to describe a partial promoter sequence which defines the start site of transcription for the linked sequence to be transcribed but which by itself is not capable of initiating transcription efficiently, if at all.

Thus, the activity of such a minimal promoter is dependent upon the binding of a transcriptional activator (such as the tetracycline-inducible fusion protein of the invention) to an operatively linked regulatory sequence (such as one or more tet operator sequences). In -23one embodiment, the minimal promoter is from the human cytomegalovirus (as described in Boshart et al. (1985) Cell 41:521-530). Preferably, nucleotide positions between about to -53 and +75 to -31 are used. Other suitable minimal promoters are known in the art or can be identified by standard techniques. For example, a functional promoter which activates transcription of a contiguously linked reporter gene chloramphenicol acetyl transferase, p-galactosidase or luciferase) can be progressively deleted until it no longer activates expression of the reporter gene alone but rather requires the presence of an additional regulatory sequence(s).

Within a transcription unit, the target nucleotide sequence (including the transcribed nucleotide. sequence and its upstream minimal promoter sequence) is operatively linked to at least one tet operator sequence. In a typical configuration, the tet operator sequence(s) is Soperatively linked upstream of the minimal promoter sequence through a phosphodiester bond at a suitable distance to allow for transcription of the target nucleotide sequence upon binding of a regulatory protein the transactivator fusion protein) to the S* 15 tet operator sequence. That is, the transcription unit is comprised of, in a 5' to 3' direction: tet operator sequence(s) a minimal promoter a transcribed nucleotide sequence. It will be appreciated by those skilled in the art that there is some flexibility in the permissable distance between the tet operator sequence(s) and the minimal promoter, although typically the tet operator sequences will be located within about 200-400 base pairs upstream of the minimal 20 promoter.

The nucleotide sequences of examples of tet-regulated promoters, containing tet operator sequences linked to a minimal promoter, that can be used in the invention are shown in SEQ ID NO: 8-10. The nucleotide sequences of SEQ ID NOs: 8 and 9 comprise a cytomegalovirus minimal promoter linked to ten tet operator sequences; the two nucleotide 25 sequences differ in the distance between the operators and the first transcribed nucleotide.

The nucleotide sequence of SEQ ID NO: 10 comprises a herpes simplex virus minimal tk promoter linked to ten tet operator sequences. The promoter of SEQ ID NO: 8 corresponds to PhCMV*-I, described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad Sci. USA 89:5547-5551. The promoter of SEQ ID NO: 9 corresponds to PhCMV*-2, also described in Gossen, M. and Bujard, H, cited supra.

Alternatively, since regulatory elements have been observed in the art to function downstream of sequences to be transcribed, it is likely that the tet operator sequence(s) can be operatively linked downstream of the transcribed nucleotide sequence. Thus, in this configuration, the transcription unit is comprised of, in a 5' to 3' direction: a minimal promoter a transcribed nucleotide sequence tet operator sequence(s). Again, it will be appreciated that there is likely to be some flexibility in the pennissable distance downstream at which the tet operator sequence(s) can be linked.

The term "tet operator sequence" is intended to encompass all classes of tet operators A, B, C, D and A nucleotide sequence to be transcribed can be operatively linked to -24a single let operator sequence, or for an enhanced range of regulation, it can be operatively linked to multiple tet operator sequences two, three, four, five, six, seven, eight, nine, ten or more operator sequences). In a preferred embodiment, the sequence to be transcribed is operatively linked to seven tet operator sequences.

A tet-regulated transcription unit can further be incorporated into a recombinant vector a plasmid or viral vector) by standard recombinant DNA techniques. The transcription unit, or recombinant vector in which itis contained, can be introduced into a host cell by standard transfection techniques, such as those described above. It should be appreciated that, after introduction of the transcription unit into a population of host cells, it may be necessary to select a host cell clone which exhibit low basal expression of the tet operator-linked nucleotide sequence selection for a host cell in which the transcription unit has integrated at a site that results in low basal expression of the tet operator-linked nucleotide sequence). Furthermore, a tet-regulated transcription unit can be introduced, by 1 procedures described above, into the genome of a non-human animal at an embryonic stage or into plant cells to create a transgenic or homologous recombinant organims carrying the transcription unit in some or all of its cells. Again, it should be appreciated that it may be necessary to select a transgenic or homologous organism in which there is low basal expression of the the tet operator-linked nucleotide sequence in cells of interest.

In one embodiment, the target nucleotide sequence of the tet-regulated transcription 20 unit encodes a protein of interest. Thus, upon induction of transcription of the nucleotide sequence by the transactivator of the invention and translation of the resultant mRNA, the protein of interest is produced in a host cell or animal. Alternatively, the nucleotide sequence to be transcribed can encode for an active RNA molecule, an antisense RNA molecule or ribozyme. Expression of active RNA molecules in a host cell or animal can be used to 25 regulate functions within the host prevent the production of a protein of interest by inhibiting translation of the mRNA encoding the protein).

A transactivator of the invention can be used to regulate transcription of an exogenous nucleotide sequence introduced into the host cell or animal. An "exogenous" nucleotide sequence is a nucleotide sequence which is introduced into the host cell and typically is inserted into the genome of the host. The exogenous nucleotide sequence may not be present elsewhere in the genome of the host a foreign nucleotide sequence) or may be an additional copy of a sequence which is present within the genome of the host but which is integrated at a different site in the genome. An exogenous nucleotide sequence to be transcribed and an operatively linked tet operator sequence(s) can be contained within a single nucleic acid molecule which is introduced into the host cell or animal.

Alternatively, a transactivator of the invention can be used to regulate transcription of an endogenous nucleotide sequence to which a tet operator sequence(s) has been linked. An "endogenous" nucleotide sequence is a nucleotide sequence which is present within the genome of the host. An endogenous gene can be operatively linked to a let operator sequence(s) by homologous recombination between a tetO-containing recombination vector and sequences of the endogeneous gene. For example, a homologous recombination vector can be prepared which includes at least one tet operator sequence and a miminal promoter sequence flanked at its 3' end by sequences representing the coding region of the endogenous gene and flanked at its 5' end by sequences from the upstream region of the endogenous gene by excluding the actual promoter region of the endogenous gene. The flanking sequences are of sufficient length for successful homologous recombination of the vector DNA with the endogenous gene. Preferably, several kilobases of flanking DNA are included in the homologous recombination vector. Upon homologous recombination between the vector DNA and the endogenous gene in a host cell, a region of the endogenous promoter is replaced by the vector DNA containing one or more tet operator sequences operably linked to a minimal promoter. Thus, expression of the endogenous gene is no longer under the control of its endogenous promoter but rather is placed under the control of the tet operator sequence(s) and the minimal promoter.

15 In another embodiment, tet operator sequences can be inserted elsewhere within an endogenous gene, preferably within a 5'or 3' regulatory region, via homologous recombination to create an endogenous gene whose expression can be regulated by a tetracycline-regulated fusion protein described herein. For example, one or more tetO sequences can be inserted into a promoter or enhancer region of an endogenous gene such 20 that promoter or enhancer function is maintained the tetO sequences are introduced into a site of the promoter/enhancer region that is not critical for promoter/enhancer function).

Regions within promoters or enhancers which can be altered without loss of promoter/enhancer function are known in the art for many genes or can be determined by standard techniques for analyzing critical regulatory regions. An endogenous gene having 25 tetO sequences inserted into a non-critical regulatory region will retain the ability to be expressed in its normal constitutive and/or tissue-specific manner but, additionally, can be downregulated by a tetracycline-controlled transcriptional inhibitor protein in a controlled manner. For example, constitutive expression of such a modified endogenous gene can be inhibited by in the presence of tetracycline (or analogue) using an inhibitor fusion protein that binds to tetO sequences in the presence of tetracycline (or analogue) (as described in further detail in Section IV and Section VI, Part B, below).

A. Regulation of Epression of tet Operator-Linked Nucleotide Sequences Expression of a tet operator-linked nucleotide sequences is regulated by a transactivator fusion protein of the invention. Thus, the fusion protein and the target nucleic acid are both present in a host cell or organism. The presence of both the transactivator fusion protein and the target transcription unit in the same host cell or organism can be achieved in a number of different ways. For example, a host cell can be transfected with one nucleic acid of the expression system encoding the transactivator fusion protein), stably -26transfected cells can be selected and then the transfected cells can be re-transfected (also referred to as "supertransfected") with nucleic acid corresponding to the other nucleic acid of the expression system the target nucleic acid to be transcribed). Two distinct selectable markers can be used for selection, uptake of the first nucleic acid can be selected with G418 and uptake of the second nucleic acid can be selected with hygromycin. Alternatively, a single population of cells can be transfected with nucleic acid corresponding to both components of the system. Accordingly, the invention provides a nucleic acid composition comprising: a first 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 1 polypeptide which activates transcription in eukaryotic cells; and 15 a second nucleic acid comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

In one embodiment, the two nucleic acids are two separate molecules two different vectors). In this case, a host cell is cotransfected with the two nucleic acid molecules or 20 successively transfected first with one nucleic acid molecule and then the other nucleic acid molecule. In another embodiment, the two nucleic acids are linked colinear) in the same molecule a single vector). In this case, a host cell is transfected with the single nucleic acid molecule.

The host cell may be a cell cultured in vitro or a cell present in vivo a cell targeted for gene therapy). The host cell can further be a fertilized ooctye, embryonic stem cell or any other embryonic cell used in the creation of non-human transgenic or homologous recombinant animals. Transgenic or homologous recombinant animals which comprise both nucleic acid components of the expression system can be created by introducing both nucleic acids into the same cells at an embryonic stage, or more preferably, an animal which carries one nucleic acid component of the system in its genome is mated to an animal which carries the other nucleic acid component of the system in its genome. Offspring which have inherited both nucleic acid components can then be identified by standard techniques.

B. Coordinate Regulation ofExpression of Two Nucleotide Sequences In addition to providing a system for the regulated expression of a single transcribed nucleotide sequence, the invention further permits coordinated regulation of the expression of two nucleotide sequences operatively linked to the same tet operator sequence(s).

Accordingly, another aspect of the invention pertains to a novel tet-regulated transcription unit for coordinate regulation of two genes. In this transcription unit, the same tet operator -27sequence(s) regulates the expression of two operatively linked nucleotide sequences that are transcribed in opposite directions from the common tet operator sequence(s). Accordingly, one nucleotide sequence is operatively linked to one side of the tet operator sequence the 5' end on the top strand of DNA) and the other nucleotide sequence is operatively linked to the opposite side of the tet operator sequence the 3' end on the top strand of DNA).

Additionally, it should be understood that each nucleotide sequence to be transcribed includes an operatively linked minimal promoter sequence which is located between the nucleotide sequence to be transcribed and the tet operator sequence(s).

A representative example of such a transcription unit is diagrammed schematically in Figure 6. In this vectors, the two nucleotide sequences, operatively linked to the same tet operator sequence(s), are transcribed in opposite directions relative to the tet operator sequence(s) the sequences are transcribed in a divergent manner upon activation by a transactivator fusion protein of the invention). By "transcribed in opposite directions relative *to the tet operator sequence(s)", it is meant that the first nucleotide sequence is transcribed 15 to 3' from one strand of the DNA the bottom strand) and the second nucleotide i" sequence is transcribed 5' to 3' from the other stand of the DNA the top strand), resulting in bidirectional transcription away from the tet operator sequence(s).

Accordingly, the invention provides a recombinant vector for coordinately-regulated, bidirectional transcription of two nucleotide sequence. In one embodiment, the vector 20 comprises a nucleotide sequence linked by phosphodiester bonds comprising, in a 5' to 3' direction: a first nucleotide sequence to be transcribed, operatively linked to at least one tet operator sequence, operatively linked to 25 a second nucleotide sequence to be transcribed, wherein transcription of the first and second nucleotide sequences proceeds in opposite directions from the at least one tet operator sequence(s) the first and second nucleotide sequences are transcribed in a divergent manner).

In another embodiment, the vector does not include the first and second nucleotide sequence to be transcribed but instead contains cloning sites which allow for the introduction into the vector of nucleotide sequences of interest. Accordingly, in this embodiment, the vector comprises a nucleotide sequence comprising in a 5' to 3' direction: a first cloning site for introduction of a first nucleotide sequence to be transcribed, operatively linked to at least one tet operator sequence, operatively linked to a second cloning site for introduction of a second nucleotide sequence to be transcribed, 28wherein transcription of a first and second nucleotide sequence introduced into the vector proceeds in opposite directions from the at least one tet operator sequence(s). It will be appreciated by those skilled in the art that this type of "cloning vector" may be in a form which also includes minimal promoter sequences such that a first nucleotide sequence introduced into the first cloning site is operatively linked to a first minimal promoter and a second nucleotide sequence introduced into the second cloning site is operatively linked to a second minimal promoter. Alternatively, the "cloning vector" may be in a form which does notinclude minimal promoter sequences and instead, nucleotide sequences including linked minimal promoter sequences are introduced into the cloning sites of the vector.

The term "cloning site" is intended to encompass at least one restriction endonuclease site. Typically, multiple different restriction endonuclease sites a polylinker) are contained within the nucleic acid.

In yet another embodiment, the vector for coordinate, bidirectional transcription of 5 two nucleotide sequences may contain a first nucleotide to be transcribed, such as that encoding a detectable marker luciferase or p-galactosidase), and a cloning site for introduction of a second nucleotide sequence of interest.

The nucleotide sequences of two different suitable bidirectional promoter regions for use in a vector for coordinate regulation of two nucleotide sequences to be transcribed, as 20 described herein, are shown in Figures 7A and 7B (SEQ ID NOS: 6 and 7, respectively). In the construct of Figure 7A, both minimal promoters present in the construct are derived from a CMV promoter. In the construct of Figure 7B, one minimal promoter present in the construct is derived from a CMV promoter, whereas the second minimal promoter is derived from a TK promoter. A plasmid pUHDG1316-8, comprising a bidirectional promoter of the invention, has been deposited on July 8, 1994 under the provisions of the Budapest Treaty at the Deutsche Sammlung Von Mikroorganismen und ZellKulturen GmbH (DSM) in o **Braunschweig, Germany and assigned deposit number DSM 9281.

The transcription unit of the invention for bidirectional transcription of two nucleotide sequences operatively linked to the same tet operator sequence(s) is useful for coordinating the expression of the two nucleotide sequences of interest. Preferably, at least one of the nucleotide sequences to be transcribed is a eukaryotic nucleotide sequence. In one application, the vector is used to produce stoichiometric amounts of two subunits of a heterodimeric molecule in the same cell. For example, the vector can be used produce antibody heavy and light chains in the same cell or to produce growth factor receptor subunits in the same cells. In another application, the vector is used to express two gene products that cooperate in establishing a particular cellular phenotype. In yet another application, the vector is used to coexpress an indicator function and a gene of interest, wherein the indicator is utilized to monitor expression of the gene of interest. Thus, one of the two coordinately expressed sequences can encode a gene of interest and the other can encode a detectable -29marker, such as a surface marker or enzyme P-galactosidase or luciferase) which is used for selection of cells expressing the gene of interest Transcription of the two coordinately-regulated nucleotide sequences can be induced by tetracycline (or an analogue thereof) by use of the Tc-inducible transcriptional activator of the invention to regulate expression of the two nucleotide sequences. Thus, in this system, expression of both nucleotide sequences is "off" in the absence ofTc (or analogue), whereas expression is turned "on" by the presence of Tc (or analogue). Alternatively, the vector for coordinate regulation of two nucleotide sequences can be used in conjunction with other tetracycline-regulated transcription factors known in the art. For example, a transactivator fusion protein of a wild-type Tet repressor fused to a transcriptional activation domain, which activates gene expression in the absence of Tc (or analogue), such as the tTA described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 82:5547-5551, can also be used in conjuction with this target transcription unit for coordinate regulation.

15 C. Independent Regulation ofExpression ofMultiple Nucleotide Sequences i* The invention still further permits independent and opposite regulation of two or more nucleotide sequences to be transcribed. Accordingly, another aspect of the invention pertains to a novel tet-regulated transcription unit for independent regulation of two or more genes. To independently regulate the expression of two nucleotide sequences to be S: 20 transcribed, one nucleotide sequence is operatively linked to a let operator sequence(s) of one class type and the other nucleotide sequence is operatively linked to a let operator sequence(s) of another class type. Accordingly, the invention provides at least one recombinant vector for independent regulation of transcription of two nucleotide sequences.

In one embodiment, the vector(s) comprises: a first nucleotide sequence to be a transcribed operatively linked to at least one *tet operator sequence of a first class type; and S a second nucleotide sequence to be a transcribed operatively linked to at least one tet operator sequence of a second class type.

(It should be understood that each nucleotide sequence to be transcribed also includes an operatively linked, upstream minimal promoter sequence.) The two independently regulated transcription units can be included on a single vector, or alternatively, on two seperate vectors. The recombinant vector(s) containing the nucleotide sequences to be transcribed can be introduced into a host cell or animal as described previously.

In another embodiment, the vector(s) does not include the first and second nucleotide sequence to be transcribed but instead contains cloning sites which allow for the introduction into the vector of nucleotide sequences of interest. Accordingly, in this embodiment, the vector(s) comprises: 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 ter operator sequence of a second class type.

This cloning vector(s) may be in a form that already includes first and second minimal promoters operatively linked, respectively, to the first and second cloning sites.

Alternatively, nucleotide sequences to be transcribed which include an operatively linked minimal promoter can be introduced into the cloning vector.

In yet another embodiment, the vector for independent regulation of two nucleotide sequences may contain a first nucleotide to be transcribed, such as that encoding a detectable marker or a suicide gene, operatively linked to at least one tet operator sequence of a first class type and a cloning site for introduction of a second nucleotide sequence of interest such S* i that it is operatively linked to at least one tet operator sequence of a second class type.

It will be appreciated by those skilled in the art that various combinations of classes of tet operator sequences can be used for independent regulation of two nucleotide sequences.

.For example, the first tet operator sequence(s) can be of the class A type and the second can *20 be of the class B type, or the first tet operator sequence can be of the class B type and the second can be of the class C type, etc. Preferably, one to the two tet operators used is a class B type operator.

Independent transcription of the first and second nucleotide sequences is regulated in a host cell by further introducing into the host cell one or more nucleic acids encoding two 25 different transactivator fusion proteins which bind independently to tet operator sequences of different class types. The first fusion protein comprises a polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue, operatively linked to a polypeptide which activates transcription in eukaryotic cells a transactivator fusion protein of the invention, such as a mutated Tnl -derived Tet repressor linked to a VP16 activation region). The second fusion protein comprises a polypeptide which binds to a tet operator sequence in the absence of tetracycline or a tetracycline analogue, operatively linked to a polypeptide which activates transcription in eukaryotic cells a wild-type TnlO-derived Tet repressor linked to a VP16 activation region, such as the tTA described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad Sci. USA 92:5547-5551). In one embodiment, the first fusion protein binds to the tet operator sequence of the first class type used in the transcription unit and the second fusion protein binds to the tet operator sequence of the second class type used in the transcription unit. Alternatively, in another embodiment, the first fusion protein binds to the second class type of tet operator and the second fusion protein binds to the first class type of tet operator.

-31- For example, the first nucleotide sequence to be transcribed may be linked to a class A tet operator and the first fusion protein may bind to class A operators, whereas the second nucleotide sequence to be transcribed may be linked to a class B tet operator and the second fusion protein may bind to class B operators. Thus, in this embodiment, transcription of the first nucleotide sequence is activated in the presence ofTc(or analogue thereof) while transcription of the second nucleotide sequence is activated in the absence of Tc (or analogue thereof). Alternatively, in another embodiment, the first fusion protein binds to class B operators and the second fusion protein binds to class A operators. In this case, transcription of the second nucleotide sequence is activated in the presence of Tc (or analogue thereof) while transcription of the first nucleotide sequence is activated in the absence of Tc (or analogue thereof). Appropriate transactivator proteins for use in this system can be designed as described above in Section I and in Gossen and Bujard (1992) cited supra. In order to inhibit heterodimerization between the two different types of Tet repressor fusion proteins present in the same cell, it may be necessary to mutate the dimerization region of one or both of the transactivator fusion proteins. Mutations can be targeted to the C-terminal region of TetR known to be involved in dimerization. The dimerization region has been described in detail based upon the crystal structure of TetR (see Hinrichs, W. et al. (1994) Science 264:418-420).

This system allows for independent and opposite regulation of the expression of two S: 20 genes by Tc and analogues thereof. Use of different Tc analogues as inducing agents may further allow for high, low or intermediate levels of expression of the different sequences (discussed in greater detail in Section V below). The novel transcription unit of the invention for independently regulating the expression of two genes, described above, can be used in situations where two gene products are to be expressed in the same cell but where it is 25 desirable to express one gene product while expression of the other gene product is turned S"off', and vice versa. For example, this system is particularly useful for expressing in the same host cell either a therapeutic gene or a suicide gene a gene which encodes a product that can be used to destroy the cell, such as ricin or herpes simplex virus thymidine kinase). In many gene therapy situations, it is desirable to be able to express a gene for therapeutic purposes in a host cell but also to have the capacity to destroy the host cell once the therapy is completed. This can be accomplished using the above-described system by linking the therapeutic gene to one class of ter operator and the suicide gene to another class of ret operator. Thus, expression of the therapeutic gene in a host cell can be stimulated by Tc (in which case expression of the suicide gene is absent). Then, once the therapy is complete, Tc is removed, which turns off expression of the therapeutic gene and turns on expression of the suicide gene in the cell.

-32- D. Combined Coordinate and Independent Regulation ofMultiple Nucleotide Sequences It is further possible to regulate the expression of four nucleotide sequences by combining the system described in Section IIIB with the system described in Section mC such that two pairs of sequences are coordinately regulated while one pair is independently regulated from the other pair. Accordingly, two target transcription units can be designed comprising: S a first nucleic acid comprising in a 5' to 3' direction: a first nucleotide sequence to be transcribed, a tet operator sequence(s) of a first class type, and a second nucleotide sequence to be transcribed a second nucleic acid comprising in a 5' to 3' direction: a third nucleotide sequence to transcribed, a tet operator sequence(s) of a second class type, and a fourth nucleotide sequence to be transcribed.

Transcription of the first and second nucleotide sequences in the first nucleic acid proceeds in a divergent manner from the first class of tet operator sequence(s). Likewise, transcription of the third and fourth nucleotide sequences in the second nucleic acide proceeds in a divergent manner from the second class of tet operator sequence(s). Thus, expression of the first and second nucleotide sequences is coordinately regulated and expression of the third and fourth nucleotide sequences is coordinately regulated. However, expression of the first and second sequences is independently (and oppositely) regulated compared to the third and fourth sequences through the use of two different transactivator fusion proteins, as described above, one which activates transcription in the presence ofTc (or analogue thereof) and the other 25 which activates transcription in the absence of Tc (or analogue thereof). One transactivator is designed to bind to a tet operators of the first class type and the other is designed to bind to a tet operators of the second class type. In other embodiments, rather than already containing first, second, third and/or fourth nucleotide sequences to be transcribed, these transcription units can contain cloning sites which allow for the introduction of first, second, third and/or fourth nucleotide sequences to be transcribed.

IV. Tetracycline-Regulated Transcriptional Inhibitors Another aspect of the invention pertains to transcriptional inhibitor fusion proteins.

The inhibitor fusion proteins of the invention are constructed similarly to the transactivator fusion proteins of the invention (see Section I above) but instead of containing a polypeptide domain that stimulates transcription in eukaryotic cells, the inhibitor fusion proteins contain a polypeptide domain that inhibits transcription in eukaryotic cells. The inhibitor fusion proteins are used to downregulate the expression of genes operably linked to tetO sequences.

For example, when a tetO-linked gene is introduced into a host cell or animal, the level of -33basal, constitutive expression of the gene may vary depending upon the type of ceU or tissue in which the gene is introduced and on the site of integration of the gene. Alternatively, constitutive expression of endogenous genes into which tetO sequences have been introduced may vary depending upon the strength of additional endogenous regulatory sequences in the vicinity. The inhibitor fusion proteins described herein provide compositions that can be used to inhibit the expression of such tetO-linked genes in a controlled manner.

In one embodiment, the inhibitor fusion protein of the invention comprises a first polypeptide that binds to tet operator sequences in the absence, but not the presence, of tetracycline (Tc) or an analogue thereof operatively linked to a heterologous second 10 polypeptide that inhibits transcription in eukaryotic cells. In another embodiment, the inhibitor fusion protein comprises a first polypeptide that binds to tet operator sequences in the presence, but not the absence, of tetracycline operatively linked to a heterologous second polypeptide that inhibits transcription in eukaryotic cells. The term "heterologous" is intended to mean that the second polypeptide is derived from a different protein than the first 15 polypeptide. Like the transactivator fusion proteins, the transcriptional inhibitor fusion proteins can be prepared using standard recombinant DNA techniques as described herein.

A. The first polypeptide of the transcriptional inhibitor fusion protein "The transcriptional inhibitor fusion protein of the invention is composed, in part, of a "20 first polypeptide which binds to a tet operator sequence either in the absence, but not the presence of tetracycline or an analogue thereof, or alternatively, (ii) in the presence, but not the absence of Tc or an analogue thereof.

Preferably, in the former embodiment, the first polypeptide is a wild-type Tet repressor (which binds to tet operator sequences in the absence but not the presence of Tc).

A wild-type Tet repressor of any class A, B, C, D or E) may be used as the first polypeptide. Preferably, the wild-type Tet repressor is a Tnl 0-derived Tet repressor. The nucleotide and amino acid sequences of a wild-type Tnl 0-derived Tet repressor are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

Alternatively, in the latter embodiment, the first polypeptide is a mutated Tet repressor as described in Section 1, part A above (which binds to tet operator sequences in the presence but not the absence of Tc). A mutated Tet repressor of any class A, B, C, D or E) may be used as the first polypeptide. Preferably, the mutated Tet repressor is a TnlOderived Tet repressor having one or more amino acid substitutions at positions 71, 95, 101 and/or 102. The nucleotide and amino acid sequences of such a mutated Tnl 0-derived Tet repressor are shown in SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

B. The second polypeptide of the transcriptional inhibitor fusion protein The first polypeptide of the transcriptional inhibitor fusion protein is operatively linked to a second polypeptide which directly or indirectly inhibits transcription in eukaryotic -34cells. As described in Section I, above, to operatively link the first and second polypeptides of a fusion protein, typically nucleotide sequences encoding the first and second polypeptides are ligated to each other in-frame to create a chimeric gene encoding the fusion protein.

However, the first and second polypeptides can be operatively linked by other means that preserve the function of each polypeptide chemically crosslinked). Although the fusion proteins are typically described herein as having the first polypeptide at the amino-terminal end of the fusion protein and the second polypeptide at the carboxy-terminal end of the fusion protein, it will be appreciated by those skilled in the art that the opposite orientation the second polypeptide at the amino-terminal end and the first polypeptide at the carbuxy- 10 terminal end) is also contemplated by the invention.

Proteins and polypeptide domains within proteins which can function to inhibit transcription in eukaryotic cells have been described in the art (for reviews see, e.g., Renkawitz, R. (1990) Trends in Genetics 6:192-197; and Herschbach, B.M. and Johnson, A.D. (1993) Annu. Rev. Cell. Biol. 2:479-509). Such transcriptional inhibitor domains have 15 been referred to in the art as "silencing domains" or "repressor domains." Although the precise mechanism by which many of these polypeptide domains inhibit transcription is not known (and the invention is not intended to be limited by mechanism), there are several possible means by which repressor domains may inhibit transcription, including: 1) competitive inhibition of binding of either activator proteins or the general transcriptional machinery, 2) prevention of the activity of a DNA bound activator and 3) negative interference with the assembly of a functional preinitiation complex of the general transcription machinery. Thus, a repressor domain may have a direct inhibitory effect on the transcriptional machinery or may inhibit transcription indirectly by inhibiting the activity of activator proteins. Accordingly, the term "a polypeptide that inhibits transcription in eukaryotic cells" as used herein is intended to include polypeptides which act either directly or indirectly to inhibit transcription. As used herein, "inhibition" of transcription is intended to mean a diminution in the level or amount of transcription of a target gene compared to the level or amount of transcription prior to regulation by the transcriptional inhibitor protein.

Transcriptional inhibition may be partial or complete. The terms "silencer", "repressor" and "inhibitor" are used interchangeably herein to describe a regulatory protein, or domains thereof, that can inhibit transcription.

A transcriptional "repressor" or "silencer" domain as described herein is a polypeptide domain that retains its transcriptional repressor function when the domain is transferred to a heterologous protein. Proteins which have been demonstrated to have repressor domains that can function when transferred to a heterologous protein include the v-erbA oncogene product (Baniahmad, A. et al. (1992) EMBOJ. 11:1015-1023), the thyroid hormone receptor (Baniahmad, supra), the retinoic acid receptor (Baniahmad, supra), and the Drosophila Krueppel (Kr) protein (Licht, J.D. et al. (1990) Nature 346:76-79; Sauer, F. and Jackle, H.

(1991) Nature 351:563-566; Licht, J.D. et al. (1994) Mol. Cell. Biol. 14:4057-4066). Nonlimiting examples of other proteins which have transcriptional repressor activity in eukaryotic cells include the Drosophila homeodomain protein even-skipped (eve), the S. cerevisiae Ssn6/Tupl protein complex (see Herschbach and Johnson, supra), the yeast SIR1 protein (see Chien, et al. (1993) Cell 25:531-541), NePI (see Kohne, et al. (1993) J. Mol. Biol. 232:747- 755), the Drosophila dorsal protein (see Kirov, et al. (1994) Mol. Cell. Biol. 14:713-722; Jiang, et al. (1993) EMBO J. 12:3201-3209), TSF3 (see Chen, et al. (1993) Mol. Cell. Biol.

13:831-840), SF1 (see Targa, et al. (1992) Biochem. Biophys. Res. Comm 18&:416-423), the Drosophila hunchback protein (see Zhang, et al. (1992) Proc. Natl. Acad Sci. USA 32:7511- 7515), the Drosophila knirps protein (see Gerwin, et al. (1994) Mol. Cell. Biol. 14:7899- 10 7908), the WTI protein (Wilm's tumor gene product) (see Anant, et al. (1994) Oncogene :3113-3126; Madden et al., (1993) Oncogene 8:1713-1720), Oct-2.1 (see Lillycrop, et al.

i (1994) Mol. Cell. Biol. 14:7633-7642), the Drosophila engrailed protein (see Badiani, et al.

(1994) Genes Dev. 8:770-782; Han and Manley, (1993) EMBO J. 12:2723-2733), E4BP4 (see Cowell and Hurst, (1994) Nucleic Acids Res. 22:59-65) and ZF5 (see Numoto, et al. (1993) 15 Nucleic Acids Res. 21:3767-3775), In a preferred embodiment, the second polypeptide of the transcriptional inhibitor fusion protein of the invention is a transcriptional silencer domain of the Drosophila Krueppel protein. A C-terminal region having repressor activity can be used, such as amino acids 403-466 of the native protein (see Sauer, F. and Jackle, supra). This region is referred to as C64KR. The nucleotide and amino acid sequences of C64KR are shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Construction of an expression vector encoding a TetR-C64KR fusion protein is described in Example 4. Alternatively, an alaninerich amino terminal region of Kr that also has repressor activity can be used as the second polypeptide of the fusion protein. For example, amino acids 26-110 of Kr (see Licht, J.D. et al., (1990) supra) can be used as the second polypeptide. Alternatively, shorter or longer polypeptide fragments encompassing either of the Kr silencer domains that still retain full or partial inhibitor activity are also contemplated amino acids 62 to 92 of the N-terminal silencer domain; see Licht, et al. (1994) supra).

In another preferred embodiment, the second polypeptide of the transcriptional inhibitor fusion protein of the invention is a transcriptional silencer domain of the v-erbA oncogene product The silencer domain of v-erbA has been mapped to approximately amino acid residues 362-632 of the native v-erbA oncogene product (see Baniahmad, et al. supra).

Accordingly, a fragment encompassing this region is used as the second polypeptide of the silencer domain. In one embodiment, amino acid residues 364-635 of the native v-erbA protein are used. The nucleotide and amino acid sequences of this region ofv-erbA are shown in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. Construction of an expression vector encoding a TetR-v-erbA fusion protein is described in Example 5. Alternatively, shorter or longer polypeptide fragments encompassing the v-erbA silencer region that still retain full or partial inhibitor activity are also contemplated. For example, a.a. residues 346- -36- 639, 362-639, 346-632, 346-616 and 362-616 of v-erbA may be used. Additionally, polypeptide fragments encompassing these regions that have internal deletions yet still retain full or partial inhibitor activity are encompassed by the invention, such as aa. residues 362- 468/508-639 of v-erbA. Furthermore, two or more copies of the silencer domain may be included in the fusion protein, such as two copies of a.a.residues 362-616 of v-erbA.

Suitable silencer polypeptide domains ofv-erbA are described further in Baniahmad, A. et al.

(supra).

In other embodiments, other silencer domains are used. Non-limiting examples of polypeptide domains that can be used include: amino acid residues 120-410 of the thyroid 10 hormone receptor alpha (THRa), amino acid residues 143-403 of the retinoic acid receptor alpha (RARa), amino acid residues 186-232 of knirps, the N-terminal region of WT 1 (see Anant, supra), the N-terminal region of Oct-2.1 (see Lillycrop, supra), a 65 amino acid domain of E4BP4 (see Cowell and Hurst, supra) and the N-terminal zinc finger domain of (see Numoto, supra). Moreover, shorter or longer polypeptide fragments encompassing 15 these regions that still retain full or partial inhibitor activity are also contemplated.

In addition to previously described transcriptional inhibitor domains, novel Stranscriptional inhibitor domains, which can be identified by standard techniques, are within the scope of the invention. The transcriptional inhibitor ability of a polypeptide can be assayed by: 1) constructing an expression vector that encodes the test silencer polypeptide linked to another polypeptide having DNA binding activity constructing a DNA binding domain-silencer domain fusion protein), 2) cotransfecting this expression vector into host cells together with a reporter gene construct that is normally constitutively expressed in the host cell and also contains binding sites for the DNA binding domain and 3) determining the amount of transcription of the reporter gene construct that is inhibited by expression of the fusion protein in the host cell. For example, a standard assay used in the art utilizes a fusion protein of a GAL4 DNA binding domain amino acid residues 1-147) and a test silencer domain. This fusion protein is then used to inhibit expression of a reporter gene construct that contains positive regulatory sequences (that normally stimulate constitutive transcription) and GAL4 binding sites (see Baniahmad, supra).

C. A third polypeptide of the transcriptional inhibitor fusion protein In addition to a Tet repressor and a transcriptional silencer domain, a transcriptional inhibitor fusion protein of the invention can contain an operatively linked third polypeptide which promotes transport of the fusion protein to a cell nucleus. As described for the transactivator fusion proteins (see Section I, Part C, above), a nuclear localization signal can be incorporated into the transcriptional inhibitor fusion protein.

-37- D. Expression of the transcriptional inhibitor fuion protein A nucleic acid molecule encoding a transcriptional inhibitor fusion protein of the invention can be incorporated into a recombinant expression vector and introduced into a host cell to express the fusion protein in the host cell as described in Section I, Parts A, B and C, above. Preferably, a host cell expressing a transcriptional inhibitor fusion protein of the invention also carries a tet operator-linked gene of interest target nucleotide sequence to be transcribed).

Transgenic organisms expressing a transcriptional inhibitor fusion protein in cells thereof can be prepared as described in Section II, Part D, above. Moreover, homologous 10 recombinant organisms expressing a transcriptional inhibitor fusion protein in cells thereof are also encompassed by the invention and can be prepared as described in Section II, Part E, above. The invention provides recombinant expression vectors suitable for homologous recombination. In one embodiment, such an expression vector comprises a nucleic acid molecule encoding a transcriptional inhibitor fusion protein of the invention which is flanked 15 at its 5' and 3' ends by additional nucleic acid of a eukaryotic gene, the additional nucleic acid *being of sufficient length for successful homologous recombination with the eukaryotic gene.

Vectors and methods for creating homologous recombinant organisms that express the components of the regulatory system of the invention, and uses therefor, are described in further detail in U.S. Patent Application Serial Number 08/260,452. Preferably, a transgenic 20 or homologous recombinant organism of the invention expressing a transcriptional inhibitor fusion protein in cells thereof also carries a tet operator-linked gene of interest target nucleotide sequence to be transcribed) in cells thereof.

V. Kits of the Invention Another aspect of the invention pertains to kits which include the components of the inducible regulatory system of the invention. Such a kit can be used to regulate the expression of a gene of interest a nucleotide sequence of interest to be transcribed) which can be cloned into a target transcription unit. The kit may include nucleic acid encoding a transcriptional activator fusion protein or a transcriptional inhibitor fusion protein or both. Alternatively, eukaryotic cells which have nucleic acid encoding a transactivator and/or inhibitor fusion protein stably incorporated therein, such that the transactivator and/or inhibitor fusion protein are expressed in the eukaryotic cell, may be provided in the kit.

In one embodiment, the kit includes a carrier means having in close confinement therein at least two container means: a first container means which contains a first nucleic acid DNA) encoding a transactivator fusion protein of the invention a recombinant expression vector encoding a first polypeptide which binds to a let operator sequence in the presence of tetracycline operatively linked to a second polypeptide which activates transcription in eukaryotic cells), and a second container means which contains a second target nucleic acid DNA) for the transactivator into which a nucleotide sequence of -38interest can be cloned. The second nucleic acid typically comprises a cloning site for introduction of a nucleotide sequence to be transcribed (optionally including an operatively linked minimal promoter sequence) and at least one operatively linked tet operator sequence.

The term "cloning site" is intended to encompass at least one restriction endonuclease site.

Typically, multiple different restriction endonuclease sites a polylinker) are contained within the nucleic acid.

To regulate expression of a nucleotide sequence of interest using the components of the kit, the nucleotide sequence is cloned into the cloning site of the target vector of the kit by S1". conventional recombinant DNA techniques and then the first and second nucleic acids are 10 introduced into a host cell or animal. The transactivator fusion protein expressed in the host cell or animal then regulates transcription of the nucleotide sequence of interest in the presence of the inducing agent (Tc or analogue thereof).

Alternatively, in another embodiment, the kit includes a eukaryotic cell which is stably transfected with a nucleic acid encoding a transactivator fusion protein of the invention 15 such that the transactivator is expressed in the cell. Thus, rather than containing nucleic acid S" alone, the first container means described above can contain a eukaryotic cell line into which the first nucleic acid encoding the transactivator has been stably introduced by stable transfection by a conventional method such as calcium phosphate precipitation or electroporation, etc.). In this embodiment, a nucleotide sequence of interest is cloned into the 20 cloning site of the target vector of the kit and then the target vector is introduced into the eukaryotic cell expressing the transactivator fusion protein.

Alternatively or additionally, a recombinant vector of the invention for coordinate regulation of expression of two nucleotide sequences can also be incorporated into a kit of the invention. The vector can be included in the kit in a form that allows for introduction into the vector of two nucleotide sequences of interest. Thus, in another embodiment, a kit of the invention includes 1) a first nucleic acid encoding a transactivator fusion protein of the invention (or a eukaryotic cell into which the nucleic acid has been stably introduced) and 2) a second nucleic acid comprising a nucleotide sequence comprising in a 5' to 3' direction: a first cloning site for introduction of a first nucleotide sequence of interest operatively linked to at least one tet operator sequence operatively linked to a second cloning site for introduction of a second nucleotide sequence of interest, wherein transcription of the first and second nucleotide sequences proceeds in opposite directions from the at least one tet operator sequence. Optionally, the vector can include operatively linked minimal promoter sequences.

In another embodiment, the vector can be in a form that already contains one nucleotide sequence to be transcribed encoding a detectable marker such as luciferase, Pgalactosidase or CAT) and a cloning site for introduction of a second nucleotide sequence of interest to be transcribed.

The transcription units and transactivators of the invention for independent regulation of expression of two nucleotide sequences to be transcribed can also be incorporated into a kit 39of the invention. The target transcription units can be in a form which allows for introduction into the transcription units of nucleotide sequences of interest to be transcribed. Thus, in another embodiment, a kit of the invention includes 1) a first nucleic acid encoding a transactivator which binds to a tet operator of a first class type in the presence of Tc or an analogue thereof, 2) 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 of a first class type, 3) a third nucleic acid encoding a transactivator which binds to a tet operator of a second class type in the absence ofTc or an analogue thereof and 4) a fourth nucleic acid comprising a second cloning site for introduction of a second nucleotide sequence to be 10 transcribed operatively linked to at least one tet operator of a second class type. (Optionally, minimal promoter sequences are included in the second and fourth nucleic acids). In another embodiment, one nucleotide sequence to be transcribed encoding a suicide gene) is already contained in either the second or the fourth nucleic acid. In yet another embodiment, the nucleic acids encoding the transactivators the first and third nucleic acids described 15 above) can be stably introduced into a eukaryotic cell line which is provided in the kit.

In yet another embodiment, a kit of the invention includes a first container means containing a first nucleic acid encoding a transcriptional inhibitor fusion protein of the invention the fusion protein inhibits transcription in eukaryotic cells either only in the presence of Tc or only the absence of Tc) 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. The kit may further include a third nucleic acid encoding a transactivator fusion protein that binds to tetO sequences either only in the presence of Tc or only in the absence of Tc. Alternatively, the first and/or third nucleic acids encoding the inhibitor or transactivator fusion proteins) may be stably incorporated into a eukaryotic host cell which is provided in the kit.

In still another embodiment, a kit of the invention may include at least one tetracycline or tetracycline analogue. For example, the kit may include a container means which contains tetracycline, anhydrotetracycline, doxycycline, epioxytetracycline or other tetracycline analogue described herein.

VI. Regulation of Gene Expression by Tetracycline or Analogues Thereof A. Stimulation of Gene Expression by Transactivator Fusion Proteins In a host cell which carries nucleic acid encoding a transactivator fusion protein of the invention and a nucleotide sequence operatively linked to the tet operator sequence(i.e., gene of interest to be transcribed), high level transcription of the nucleotide sequence operatively linked to the tet operator sequence(s) does not occur in the absence of the inducing agent, tetracycline or analogues thereof. The level of basal transcription of the nucleotide sequence may vary depending upon the host cell and site of integration of the sequence, but is generally quite low or even undetectable in the absence of Tc. In order to induce transcription in a host cell, the host cell is contacted with tetracycline or a tetracycline analogue. Accordingly, another aspect of the invention pertains to methods for stimulating transcription of a nucleotide sequence operatively linked to a tet operator sequence in a host cell or animal which expresses a transactivator fusion protein of the invention. The methods involve contacting the cell with tetracycline or a tetracycline analogue or administering tetracycline or a tetracycline analogue to a subject containing the cell.

The term "tetracycline analogue" is intended to include compounds which are structurally related to tetracycline and which bind to the Tet repressor with a K, of at least 10 about 10 6

M-

1 Preferably, the tetracycline analogue binds with an affinity of about 10 9

M-

1 or greater. Examples of such tetracycline analogues include, but are not limited to, anhydrotetracycline, doxycycline, chlorotetracycline, oxytetracycline and others disclosed by Hlavka and Boothe, "The Tetracyclines," in Handbook of Experimental Pharmacology 78, R.K. Blackwood et al. Springer-Verlag, Berlin-New York, 1985; L.A. Mitscher, "The 15 Chemistry of the Tetracycline Antibiotics", Medicinal Research 9, Dekker, New York, 1978; Noyee Development Corporation, "Tetracycline Manufacturing Processes" Chemical Process Reviews, Park Ridge, NJ, 2 volumes, 1969; R.C. Evans, "The Technology of the Tetracyclines", Biochemical Reference Series 1, Quadrangle Press, New York, 1968; and H.F. Dowling, "Tetracycline", Antibiotic Monographs, no. 3, Medical Encyclopedia, New York, 1955. Preferred Tc analogues for high level stimulation of transcription are anhydrotetracycline and doxycycline. A Tc analogue can be chosen which has reduced antibiotic activity compared to Tc. Examples of such Tc analogues are anhydrotetracycline, epioxytetracycline and cyanotetracycline.

To induce gene expression in a cell in vitro, the cell is contacted with Tc or a Tc analogue by culturing the cell in a medium containing the compound. When culturing cells in vitro in the presence of Tc or Tc analogue, a preferred concentration range for the inducing agent is between about 10 and about 1000 ng/ml. Tc or a Tc analogue can be directly added to media in which cells are already being cultured, or more preferably for high levels of gene induction, cells are harvested from Tc-free media and cultured in fresh media containing Tc, or an analogue thereof.

To induce gene expression in vivo, cells within in a subject are contacted with Tc or a Tc analogue by administering the compound to the subject. The term "subject" is intended to include humans and other non-human mammals including monkeys, cows, goats, sheep, dogs, cats, rabbits, rats, mice, and transgenic and homologous recombinant species thereof.

Furthermore, the term "subject" is intended to include plants, such as transgenic plants.

When the inducing agent is administered to a human or animal subject, the dosage is adjusted to preferably achieve a serum concentration between about 0.05 and 1.0 tg/ml. Tc or a Tc analogue can be administered to a subject by any means effective for achieving an in vivo concentration sufficient for gene induction. Examples of suitable modes of administration -41include oral administration dissolving the inducing agent in the drinking water), slow release pellets and implantation of a diffusion pump. To administer Tc or a Tc analogue to a transgenic plant, the inducing agent can be dissolved in water administered to the plant.

The ability to use different Tc analogues as inducing agents in this system allows for modulate the level of expression of a tet operator-linked nucleotide sequence. As demonstrated in Example 2, anhydrotetracycline and doxycycline have been found to be strong inducing agents. The increase in transcription of the target sequence is typically as high as 1000- to 2000-fold, and induction factors as high as 20,000 fold can be achieved.

Tetracycline, chlorotetracycline and oxytetracycline have been found to be weaker inducing 10 agents, the increase in transcription of a target sequence is in the range of about *Thus, an appropriate tetracycline analogue is chosen as an inducing agent based upon the desired level of induction of gene expression. It is also possible to change the level of gene expression in a host cell or animal over time by changing the Tc analogue used as the inducing agent. For example, there may be situations where it is desirable to have a strong 15 burst of gene expression initially and then have a sustained lower level of gene expression.

Accordingly, an analogue which stimulates a high levels of transcription can be used initially as the inducing agent and then the inducing agent can be switched to an analogue which stimulates a lower level of transcription. Moreover, when regulating the expression of multiple nucleotide sequences when one sequence is regulated by a one of class tet operator sequence(s) and the other is regulated by another class of ret operator sequence(s), as described above in Section III, Part C, above), it may be possible to independently vary the level of expression of each sequence depending upon which transactivator fusion protein is *i used to regulate transcription and which Tc analogue(s) is used as the inducing agent.

Different transactivator fusion proteins are likely to exhibit different levels of responsiveness to Tc analogues. The level of induction of gene expression by a particular combination of transactivator fusion protein and inducing agent (Tc or Tc analogue) can be determined by techniques described herein, see Example Additionally, the level of gene expression can be modulated by varying the concentration of the inducing agent. Thus, the expression system of the invention provides a mechanism not only for turning gene expression on or off, but also for "fine tuning" the level of gene expression at intermediate levels depending upon the type and concentration of inducing agent used.

B. Inhibition of Gene Expression by Transcriptional Inhibitor Fusion Proteins The invention also provides methods for inhibiting gene expression using the transcriptional inhibitor fusion proteins of the invention. These methods can be used to down-regulate basal, constitutive or tissue-specific transcription of a retO-linked gene of interest For example, a gene of interest that is operatively linked to tetO sequences and additional positive regulatory elements consitutive or tissue-specific enhancer sequences) will be transcribed in host cells at a level that is primarily determined by the -42strength of the positive regulatory elements in the host cell. Moreover, a gene of interest that is operatively linked to tetOsequences and only a minimal promoter sequence may exhibit varying degrees of basal level transcription depending on the host cell or tissue and/or the site of integration of the sequence. In a host cell containing such a target sequence and expressing an inhibitor fusion protein of the invention, transcription of the target sequence can be down regulated in a controlled manner by altering the concentration of Tc (or analogue) in contact with the host cell. For example, when the inhibitor fusion protein binds to tetO in the absence of Tc, the concentration of Tc in contact with the host cell is reduced to inhibit expression of the target gene. Preferably, a host cell is cultured in the absence of Tc to 10 keep target gene expression repressed. Likewise, Tc is not administered to a host organism to keep target gene expression repressed. Alternatively, when the inhibitor fusion protein binds i to tetO in the presence of Tc, the concentration of Tc in contact with the host cell is increased to inhibit expression of the target gene. For example, Tc is added to the culture medium of a host cell or Tc is administered to a host organism to repress target gene expression.

15 The inhibitor fusion proteins described herein can inhibit a tetO-linked gene of interest in which the tetO sequences are positioned 5' of a minimal promoter sequence tetracycline-regulated transcription units as described in Section II, above). Furthermore, the inhibitor fusion protein may be used to inhibit expression of a gene of interest in which tetOlinked sequences are located 3' of the promoter sequence but 5' of the transcription start site.

Still further, the inhibitor fusion protein may be used to inhibit expression of a gene of interest in which tetO-linked sequences are located 3' of the transcription start site.

Various Tc analogues as described in Section VI, part A, above, with respect to the transactivator fusion proteins can similarly be used to regulate the activity of the inhibitor fusion proteins. Moreover, the methods of in vitro culture with Tc (or analogue) and in vivo administration of Tc (or analogue) described in Section VI, part A, are equally applicable to the transcriptional inhibitor fusion proteins.

C. Combined Positive and Negative Regulation of Gene Expression In addition to regulating gene expression using either a transcriptional activator or inhibitor fusion protein alone, the two types of fusion proteins can be used in combination to allow for both positive and negative regulation of expression of one or more target genes in a host cell. Thus, a transcriptional inhibitor protein that binds to tetO either in the absence, but not the presence, of Tc, or (ii) in the presence, but not the absence, of Tc, can be used in combination with a transactivator protein that binds to tetO either in the absence, but not the presence, of Tc, or (ii) in the presence, but not the absence, of Tc. Transactivator proteins that bind to tetO in the absence, but not the presenc, of Tc wild-type TetR-activator fusion proteins) are described in further detail in USSN 08/076,726, USSN 08/076,327 and USSN 08/260,452. Transactivator fusion proteins that bind to tetO in the presence, but not the absence, of Tc mutated TetR-activator fusion proteins) are described herein (see -43- Section I above) and in USSN 08/270,637 and USSN 08/275,876. Transcriptional inhibitor fusion proteins are described herein in Section IV.

As described above in Section I, Part C, when more than one TetR fusion protein is expressed in a host cell or organism, additional steps may be taken to inhibit heterodimerization between the different TetR fusion proteins. For example, a transactivator composed of a TetR of one class may be used in combination with a transcriptional inhibitor composed of a TetR of a second, different class that does not heterodimerize with the first class of TetR. Alternatively, amino acid residues of the TetR involved in dimerization may be mutated to inhibit heterodimerization. However, even if some heterodimerization between 10 transactivator and inhibitor fusion proteins occurs in a host cell, sufficient amounts of homodimers should be produced to allow for efficient positive and negative regulation as described herein.

It will be appreciated by those skilled in the art that various combinations of activator and inhibitor proteins can be used to regulate a single tetO-linked gene of interest in both a 15 positive and negative manner or to regulate multiple tetO-linked genes of interest in a coordinated manner or in an independent manner using the teachings described herein. The precise regulatory components utilized will depend upon the genes to be regulated and the type of regulation desired. Several non-limiting examples of how the transactivator and inhibitor fusion proteins may be used in combination are described further below. However, many other possible combinations will be evident to the skilled artisan in view of the teachings herein and are intended to be encompassed by the invention.

In a preferred embodiment, illustrated schematically in Figure 10, expression of a i etO-linked target gene of interest in a host cell is regulated in both a negative and positive manner by the combination of an inhibitor fusion protein that binds to tetO in the absence, but not the presence, of tetracycline or analogue thereof (referred to as a tetracycline controlled silencing domain, or tSD) and an activator fusion protein that binds to tetO in the presence, but not the absence, of tetracycline or analogue thereof (referred to as a reverse tetracycline controlled transactivator, or rtTA). In addition to tetO sequences, the target gene is linked to a promoter, and may contain other positive regulatory elements enhancer sequences) that contribute to basal level, constitutive transcription of the gene in the host cell.

Binding of tSD to the tetO sequences in the absence of tetracycline or analogue doxycycline) inhibits the basal constitutive transcription of the gene of interest, thus keeping the gene of interest in a repressed state until gene expression is desired. When gene expression is desired, the concentration of tetracycline or analogue doxycycline) in contact with the host cell increased. Upon addition of the drug, tSD loses the ability to bind to tetO sequences whereas the previously unbound rtTA acquires the ability to bind to tetO sequences. The resultant binding of rtTA to the tetO sequences linked to the gene of interest thus stimulates transcription of the gene of interest. The level of expression may be controlled by the concentration of tetracycline or analogue, the type of Tc analogue used, the -44duration of induction, etc., as described previously herein. It will be appreciated that the reverse combination of fusion proteins (iLe., the inhibitor binds in the presence but not the absence of the drug and the activator binds in the absence but not the presence of the drug) can also be used. In this case, expression of the gene of interest is kept repressed by contacting the host cell with the drug culture with Tc- or analogue) and gene expression is activated by removal of the drug.

in another embodiment, the activator and inhibitor fusion proteins,as described in the previous paragraph, are used in combination to coordinately regulate, in both a positive and negative manner, two genes of interest using the bidirectional tetO-linked transcription unit described in Section MI, Part B above. In this case, Gene I and Gene 2 are linked to the same tei'O sequence(s), but in opposite orientations. The inhibitor fusion protein is used to repress basal levels of transcription of both Gene I and Gene2 in a coordinate manner, whereas the transactivatcr- fusion protein is used to stimuh'-e expression of Gene I and Gene 2 in a coordinate manner.

In yet another embodiment, the activator and inhibitor fusion proteins are used to independently regulate two or more genes of interest using the tetO-linked transcription units as described in Section 1I1, Part C above. For example, in one embodiment, a tr-ansactivator fusion protein that binds to one class of tetO sequences class A) in the presence, but not the absence of Tc or analogue is used in combination with an inhibitor fusion protein that binds to a second, different class of tetO sequences class B) also in the presence, but not the absence, of Tc or analogue. In a host cell containing Gene I linked to class A tetO sequences and Gene 2 linked to class B tetO sequences, both genes will be expressed at basal levels in the absence of the drug, whereas expression of Gene 1 will be stimulated upon addition of the drug arnd expression of Gene 2 will be repressed upon addition of the drug.

Alternatively, in another embodiment, the transactivator binds to one class of te1O sequences class A) in the presence, but not the absence, of Tc or analogue and the inhibitor fusion protein binds to a second, different class of tetO sequences class B) in the absence but not the presence of Tc or analogue. In the host cell as described in the previous paragraph, Gene I will be expressed at basal levels in the absence of the drug and 30 will be stimulated upon addition of the drug, whereas Gene 2 will be repressed in the absence of the drug but will have basal levels expression upon addition of the drug. Various other possible combinations will be apparent to the skilled artisan. Transactivator and inhibitor fusion proteins that bind to different classes of WeO sequences can be prepared as described in Section I, Part A. Target transcription units comprising tefO sequences of different classes can be prepared as described in Section m1, Part C.

VT. Applications of the Invention The invention is widely applicable to a variety of situations where it is desirable to be able to turn gene expression on and off, or regulate the level of gene expression, in a rapid, efficient and controlled manner without causing pleiotropic effects or cytotoxicity. Thus, the system of the invention has widespread applicability to the study of cellular development and differentiation in eukaryotic cells, plants and animals. For example, expression of oncogenes can be regulated in a controlled manner in cells to study their function. Additionally, the system can be used to regulate the expression of site-specific recombinases, such as CRE or FLP, to thereby allow for irreversible modification of the genotype of a transgenic organism under controlled conditions at a particular stage of development. For example, drug resistance markers inserted into the genome of transgenic plants that allow for selection of a particular transgenic plant could be irreversibly removed via a Tc-regulated site specific recombinase. Other applications of the regulatory system of the invention include: A. Gene Therapy The invention may be particularly useful for gene therapy purposes, in treatments for either genetic or acquired diseases. The general approach of gene therapy involves the introduction of nucleic acid into cells such that one or more gene products encoded by the 20 introduced genetic material are produced in the cells to restore or enhance a functional activity. For reviews on gene therapy approaches see Anderson, W.F. (1992) Science 256:808-813; Miller, A.D. (1992) Nature 357:455-460; Friedmann, T. (1989) Science 244:1275-1281; and Cournoyer, et al. (1990) Curr. Opin. Biotech. 1:196-208. However, current gene therapy vectors typically utilize constitutive regulatory elements which are 25 responsive to endogenous transcriptions factors. These vector systems do not allow for the ability to modulate the level of gene expression in a subject. In contrast, the inducible regulatory system of the invention provides this ability.

To use the system of the invention for gene therapy purposes, in one embodiment, cells of a subject in need of gene therapy are modified to contain 1) nucleic acid encoding a 30 transactivator fusion protein of the invention in a form suitable for expression of the transactivator in the host cells and 2) a gene of interest for therapeutic purposes) operatively linked to a tet operator sequence(s). The cells of the subject can be modified ex vivo and then introduced into the subject or the cells can be directly modified in vivo (methods for modification of the cells are described above in Section II). Expression of the gene of interest in the cells of the subject is then stimulated by administering Tc or a Tc analogue to the patient. The level of gene expression can be varied depending upon which particular Tc analogue is used as the inducing agent The level of gene expression can also be modulated by adjusting the dose of the tetracycline, or analogue thereof, administered to -46the patient to thereby adjust the concentration achieved in the circulation and the tissues of interest.

Moreover, in another embodiment, a transcriptional inhibitor fusion protein is used to further control the level of expression of the gene of interest. For example, the cells of the subject can be modified to also contain a nucleic acid encoding a transcriptional inhibitor fusion protein that binds to tetO in the absence of Tc. The nucleic acid is in a form suitable for expression of the inhibitor fusion protein in the host cells. Thus, prior to administration of Tc (or analogue) to the subject, the basal level of transcription of the gene of interest will be kept silent by the inhibitor fusion protein. Upon administration of Tc, binding of the inhibitor fusion protein to tetO will be inhibited whereas binding of the transactivator fusion will be induced, thereby stimulating transcription of the gene of interest. Such combined positive and negative regulation of gene expression using both a transactivator fusion protein and transcriptional inhibitor fusion protein of the invention is illustrated schematically in Figure Conventional detection methods known in the art, such as an enzyme linked immunosorbent assay, can be used to monitor the expression of the regulated protein of interest in the host cells and the concentration of Tc or Tc analogue can be varied until the desired level of expression of the protein of interest is achieved. Accordingly, expression of a protein of interest can be adjusted according to the medical needs of an individual, which 20 may vary throughout the lifetime of the individual. To stop expression of the gene of interest Sin cells of the subject, administration of the inducing agent is stopped. Thus, the regulatory S. system of the invention offers the advantage over constitutive regulatory systems of allowing for modulation of the level of gene expression depending upon the requirements of the therapeutic situation.

Genes of particular interest to be expressed in cells of a subject for treatment of genetic or acquired diseases include those encoding adenosine deaminase, Factor VIII, Factor IX, dystrophin, p-globin, LDL receptor, CFTR, insulin, erythropoietin, anti-angiogenesis S factors, growth hormone, glucocerebrosidase, p-glucouronidase, al-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, ornithine transcarbamylase, S. 30 arginosuccinate synthetase, UDP-glucuronysyl transferase, apoAl, TNF, soluble TNF receptor, interleukins IL-2), interferons a- or y-IFN) and other cytokines and growth factors. Cells types which can be modified for gene therapy purposes include hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, skin epithelium and airway epithelium. For further descriptions of cell types, genes and methods for gene therapy see Wilson, J.M et al. (1988) Proc. Natl. Acad Sci. USA 8J:3014-3018; Armentano, D. et al.

(1990) Proc. Natl. Acad Sci. USA 87:6141-6145; Wolff, J.A. et al. (1990) Science 242:1465- 1468; Chowdhury, J.R. et al. (1991) Science 254:1802-1805; Ferry, N. et al. (1991) Proc.

Natl. Acad Sci. USA 88:8377-8381; Wilson, J.M. et al. (1992) J. Biol. Chem. 262:963-967; Quantin, B. et al. (1992) Proc. Natl. Acad Sci. USA 28:2581-2584; Dai, Y. et al. (1992) -47- Proc. Natl. Acad Sci. USA 82:10892-10895; van Beusechem, V.W. et al. (1992) Proc. Natl.

Acad Sci. USA 28:7640-7644; Rosenfeld, M.A. et al. (1992) Cell f6:143-155; Kay, MA. et al. (1992) Human Gene Therapy 1:641-647; Cristiano, R.J. et al. (1993) Proc. Natl. Acad Sci. USA 2Q:2122-2126; Hwu, P. et al. (1993) J Immunol. 1L5:4104-4115; and Herz, J. and Gerard, R.D. (1993) Proc. Nail. Acad Sci. USA 2902812.2816.

Gene therapy applications of particular interest in cancer treatment include overexpression of a cytokine gene TNF-a) in tumor infiltrating lymphocytes or ectopic expression of cytokines in tumor cells to induce an anti-tumor immune response at the tumor site), expression of an enzyme in tumor cells which can convert a non-toxic agent into a toxic agent, expression of tumor specific antigens to induce an anti-tumor immune response, expression of tumor suppressor genes p53 or Rb) in tumor cells, expression of a multidrug resistance gene MDR1 and/or MRP) in bone marrow cells to protect them from the toxicity of chemotherapy.

Gene therapy applications of particular interest in treatment of viral diseases include expression of trans-dominant negative viral transactivation proteins, such as trans-dominant negative tat and rev mutants for HIV or trans-dominant ICp4 mutants for HSV (see e.g., Balboni, P.G. et al. (1993) J. Med. Virol. 41:289-295; Liem, S.E. et al. (1993) Hum. Gene Ther. 4:625-634; Malim, M.H. et al. (1992) J. Exp. Med 126:1197-1201; Daly, T.J. et al.

(1993) Biochemistry 32:8945-8954; and Smith, C.A. et al. (1992) Virology 121:581-588), 20 expression of trans-dominant negative envelope proteins, such as env mutants for HIV (see Steffy, K.R. et al. (1993) J. Virol. 62:1854-1859), intracellular expression of antibodies, or fragments thereof, directed to viral products ("internal immunization", see Marasco, W.A. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893) and expression of soluble viral receptors, such as soluble CD4. Additionally, the system of the invention can be used to conditionally express a suicide gene in cells, thereby allowing for elimination of the cells after they have served an intended function. For example, cells used for vaccination can be eliminated in a subject after an immune response has been generated the subject by inducing expression of a suicide gene in the cells by. administering Tc or a Tc analogue to the subject.

The Tc-controlled regulatory system of the invention has numerous advantages 30 properties that it particularly suitable for application to gene therapy. For example, the system provides an "on"/"off" switch for gene expression that allows for regulated dosaging a gene product in a subject There are several situations in which it may be desirable to be able to provide a gene product at specific levels and/or times in a regulated manner, rather than simply expressing the gene product constitutively at a set level. For example, a gene of interest can be switched "on" at fixed intervals daily, alternate days, weekly, etc.) to provide the most effective level of a gene product of interest at the most effective time. The level of gene product produced in a subject can be monitored by standard methods direct monitoring using an immunological assay such as ELISA or RIA or indirectly by monitoring of a laboratory parameter dependent upon the function of the gene product of 48interest, blood glucose levels and the like). This ability to turn "on" expression of a gene at discrete time intervals in a subject while also allowing for the gene to be kept "off" at other times avoids the need for continued administration of a gene product of interest at intermittent intervals. This approach avoids the need for repeated injections of a gene product, which may be painful and/or cause side effects and would likely require continuous visits to a physician. In contrast, the system of the invention avoids these drawbacks.

Moreover, the ability to turn "on" expression of a gene at discrete time intervals in a subject allows for focused treatment of diseases which involve "flare ups" of activity many autoimmune diseases) only at times when treatment is necessary during the acute phase when pain and symptoms are evident. At times when such diseases are in remission, the expression system can be kept in the "off' state.

Gene therapy applications that may particularly benefit from this ability to modulate gene expression during discrete time intervals include the following non-limiting examples: Rheumatoid arthritis genes which encode gene products that inhibit the production of inflammatory cytokines TNF, IL-l and IL-12). can be expressed in subjects.

Examples of such inhibitors include soluble forms of a receptor for the cytokine.

'"Additionally or alternatively, the cytokines IL-I 0 and/or IL-4 (which stimulate a protective Th2-type response) can be expressed. Moreover, a glucocorticomimetic receptor (GCMR) can be expressed.

Hyopituitarism the gene for human growth hormone can be expressed in such subjects only in early childhood, when gene expression is necessary, until normal stature is achieved, at which time gene expression can be downregulated.

•Wound healing/Tissue regeneration Factors growth factors, angiogenic factors, etc.) necessary for the healing process can be expressed only when needed and then downregulated.

,Anti-Cancer Treatments Expression of gene products useful in anti-cancer treatment can be limited to a therapeutic phase until retardation of tumor growth is achieved, at which time expression of the gene product can be downregulated. Possible systemic anti-cancer 0 treatments include use of tumor infiltrating lymphocytes which express immunostimulatory 30 molecules IL-2, IL-12 and the like), angiogenesis inhibitors (PF4, IL-12, etc.), Herregulin, Leukoregulin (see PCT Publication No. WO 85/04662), and growth factors for bone marrow support therapy, such as G-CSF, GM-CSF and M-CSF. Regarding the latter, use of the regulatory system of the invention to express factors for bone marrow support therapy allows for simplified therapeutic switching at regular intervals from chemotherapy to bone marrow support therapy (similarly, such an approach can also be applied to AIDS treatment, simplified switching from anti-viral treatments to bone marrow support treatment).

Furthermore, controlled local targeting of anti-cancer treatments are also possible. For example, expression of a suicide gene by a regulator of the invention, wherein the regulator -49itself is controlled by, for example, a tumor-specific promoter or a radiation-induced promoter.

In another embodiment, the regulatory system of the invention is used to express angiogenesis inhibitor(s) from within a tumor via a transgene regulated by the system of the invention. Expression of angiogenesis inhibitors in this manner may be more efficient than systemic administration of the inhibitor and would avoid any deleterious side effects that might accompany systemic administration. In particular, restricting angiogenesis inhibitor expression to within tumors could be particularly useful in treating cancer in children still undergoing angiogenesis associated with normal cell growth.

In another embodiment, high level regulated expression of cytokines may represent a method for focusing a patients own immune response on tumor cells. Tumor cells can be transduced to express chemoattractant and growth promoting cytokines important in increasing an individual's natural immune response. Because the highest concentrations of cytokines will be in the proximity of the tumor, the likelihood of eliciting an immunological response to tumor antigens is increased. A potential problem with this type of therapy is that those tumor cells producing the cytokines will also be targets of the immune response and "therefor the source of the cytokines will be eliminated before eradication of all tumor cells can be certain. To combat this, expression of viral proteins known to mask infected cells from the immune system can be placed under regulation, along with the cytokine gene(s), in 20 the same cells. One such protein is the E19 protein from adenovirus (see Cox, Science 247:715). This protein prevents transport of class I HLA antigens to the surface of the cell and hence prevents recognition and lysis of the cell by the host's cytotoxic T cells.

Accordingly, regulated expression of E19 in tumor cells could shield cytokine producer cells from cytotoxic T cells during the onset of an immune response provoked by cytokine expression. After a sufficient period of time has elapsed to eradicate all tumor cells but those expressing E19, El9 expression can be turned off, causing these cells then to fall victim to the provoked anti-tumor immune response.

Benin prostatic vpertrohy Similar to the above, a suicide gene can be regulated by a regulator of the invention, wherein the regulator itself is controlled by, for example, a 30 prostate-specific promoter.

The ability to express a suicide gene an apoptosis gene, TK gene, etc) in a controlled manner using the regulatory system of the invention adds to the general safety and usefulness of the system. For example, at the end of a desired therapy, expression of a suicide gene can be triggered to eliminate cells carrying the gene therapy vector, such as cells in a bioinert implant, cells that have disseminated beyond the intended original location, etc.

Moreover, if a transplant becomes tumorous or has side effects, the cells can be rapidly eliminated by induction of the suicide gene. The use of more than one Tc-controlled "on"/"off" switch in one cell allows for completely independent regulation of a suicide gene compared to regulation of a gene of therapeutic interest (as described in detail herein).

The regulatory system of the invention further offers the ability to establish a therapeutically relevant expression level for a gene product of interest in a subject, in contrast to unregulated constitutive expression which offers no flexibility in the level of gene product expression that can be achieved. A physiologically relevant level of gene product expression can be established based on the particular medical need of the subject, based on laboratory tests that monitor relevant gene product levels (using methods as described above).

In addition to the clinical examples and gene products already discussed above with gene to dosaging of the gene product, other therapeutically relevant gene products which can be expressed at a desired level at a desired time include: Factor XIII and IX in hemophiliacs iu expression can be elevated during times of risk of injury, such as during sports); insulin or amylin in diabetics (as needed, depending on the state of disease in the subject, diet, etc.); erythropoietin to treat erythrocytopenia (as needed, at end-stage renal failure); lowdensity lipoprotein receptor (LDLr) or very low-density lipoprotein receptor (VLDLr) for artherosclerosis or gene therapy in liver using ex vivo implants). Applications to treatment of central nervous system disorders are also encompassed. For example, in Alzheimer's disease, "fine tuned" expression of choline acetyl transferase (ChAT) to restore acetylcholine levels, neurotrophic factors NGF, BDNGF and the like) and/or complement inhibitors sCR1, sMCP, sDAF, sCD59 etc.) can be accomplished. Such gene products can be provided, for example, by transplanted cells expressing the gene 20 products in a regulated manner using the system of the invention. Moreover, Parkinson's disease can be treated by "fine tuned" expression of tyrosine hydroxylase (TH) to increase levodopa and dopamine levels.

In addition to the proteinaceous gene products discussed above, gene products that are S* functional RNA molecules (such as anti-sense RNAs and ribozymes) can be expressed in a 25 controlled manner in a subject for therapeutic purposes. For example, a ribozyme can be designed which discriminates between a mutated form of a gene and a wild-type gene.

Accordingly, a "correct" gene a wild-type p53 gene) can be introduced into a cell in 0* parallel with introduction of a regulated ribozyme specific for the mutated form of the gene a mutated endogenous p53 gene) to remove the defective mRNA expressed from the 30 endogenous gene. This approach is particularly advantageous in situations in which a gene product from the defective gene would interfere with the action of the exogenous wild-type gene.

Expression of a gene product in a subject using the regulatory system of the invention is modulated using tetracycline or analogues thereof. Such drugs can be administered by any route appropriate for delivery of the drug to its desired site of action delivery to cells containing a gene whose expression is to be regulated). Depending on the particular cell types involved, preferred routes of administration may include oral administration, intravenous administration and topical administration using a transdermal patch to reach -51cells of a localized transplant under the skin, such as keratinocytes, while avoiding any possible side effects from systemic treatment).

In certain gene therapy situations, it may be necessary or desirable to take steps to avoid or inhibit unwanted immune reactions in a subject receiving treatment To avoid a reaction against the cells expressing the therapeutic gene product, a subject's own cells are generally used, when possible, to express the therapeutic gene product, either by in vivo modification of the subject's cells or by obtaining cells from the subject, modifying them ex vivo and returning them to the subject. In situations where allogeneic or xenogeneic cells are used to express a gene product of interest, the regulatory system of the invention, in addition to regulating a therapeutic gene, can also be used to regulate one or more genes involved in the immune recognition of the cells to inhibit an immune reaction against the foreign cells.

For example, cell-surface molecules involved in recognition of a foreign cell by T lymphocytes can be downmodulated on the surface of a foreign cell used for delivery of a therapeutic gene product, such as by regulated expression in the foreign cell of a ribozyme which cleaves the mRNA encoding the cell-surface molecule. Particularly preferred cell surface molecules which can be downmodulated in this manner to inhibit an unwanted immune response include class I and/or class II major histocompatibility complex (MHC) molecules, costimulatory molecules B7-1 and/or B7-2), CD40, and various "adhesion" molecules, such as ICAM-I or ICAM-2. Using approaches described herein for independent 20 but coordinate regulation of multiple genes in the same cell, the down-regulation of expression of a cell-surface molecule(s) in a host cell can be coordinated with the upregulation of expression of a therapeutic gene. Accordingly, after therapy is completed and expression of the therapeutic gene is halted, expression of the endogenous cell surface molecule(s) can be restored to normal.

Furthermore, as described above regarding anti-cancer treatments, a viral protein adenovirus El9 protein) that downmodulates expression of MHC antigens can be regulated in host cells using the system of the invention as a means of avoiding unwanted immunological reactions.

In addition to avoiding or inhibiting an immune response against a foreign cell 30 delivering a therapeutic gene product, it may also be necessary, in certain situations, to avoid or inhibit an immune response against certain components of the regulatory system of the invention the regulator fusion proteins described herein) that are expressed in a subject, since these fusion proteins contain non-mammalian polypeptides that may stimulate an unwanted immune reaction. In this regard, regulator fusion proteins can be designed and/or selected for a decreased ability to stimulate an immune response in a host. For example, a transcriptional activator domain for use in the regulator fusion protein can be chosen which has minimal immunogenicity. In this regard, a wild-type transcriptional activation domain of the herpes simplex virus protein VP16 may not be a preferred transcriptional activation domain for use in vivo, since it may stimulate an immune response in mammals. Alternative -52transcriptional activation domains can be used, as described herein, based on their reduced immunogenicity in a subject For example, a transcriptional activation domain of a protein of the same species as the host may be preferred a transcriptional activation domain from a human protein for use of a regulatory fusion protein in humans). Alternatively, a regulatory fusion protein of the invention can be modified to reduce its immunogenicity in subjects, e.g., by identifying and modifying one or more dominant T cell epitopes within a polypeptide of the fusion protein either the Tet repressor moiety or the transcriptional modulator moiety, such as a VP 16 polypeptide). Such T cell epitopes can be identified by standard methods and altered by mutagenesis, again by standard methods. A modified form of a regulator fusion protein can then be selected which retains its original transcriptional regulatory ability yet which exhibits reduced immunogenicity in a subject as compared to an unmodified fusion protein.

In addition to the foregoing, all conventional methods for generally or specifically downmodulating immune responses in subjects can be combined with the use of the regulatory system of the invention in situations where inhibition of immune responses is desired. General immunosuppressive agents, such as cyclosporin A and/or FK506, can be administered to the subject Alternatively, immunomodulatory agents which may allow for o. "more specific immunosuppression can be used. Such agents may include inhibitors of ocostimulatory molecules a CTLA4Ig fusion protein, soluble CD4, anti-CD4 antibodies, 20 anti-B7-1 and/or anti-B7-2 antibodies or anti-gp39 antibodies).

Finally, in certain situations, a delivery vehicle for cells expressing a therapeutic gene can be chosen which minimizes exposure of transplanted cells to the immune system. For example, cells can be implanted into bioinert capsules/biocompatible membranes with pores S* which allow for diffusion of proteins a therapeutic gene product of interest) out of the 25 implant and diffusion of nutrients and oxygen into the implant but which prevent entry of immune cells, thereby avoiding exposure of the transplanted cells to the immune system (as S. has been applied to islet cell transplantation).

B. Production of Proteins in Vitro 30 Large scale production of a protein of interest can be accomplished using cultured cells in vitro which have been modified to contain 1) a nucleic acid encoding a transactivator fusion protein of the invention in a form suitable for expression of the transactivator in the cells and 2) a gene encoding the protein of interest operatively linked to a tet operator sequence(s). For example, mammalian, yeast or fungal cells can be modified to contain these nucleic acid components as described herein. The modified mammalian, yeast or fungal cells can then be cultured by standard fermentation techniques in the presence of Tc or an analogue thereof to induce expression of the gene and produce the protein of interest. Accordingly, the invention provides a production process for isolating a protein of interest. In the process, a host cell a yeast or fungus), into which has been introduced both a nucleic acid -53encoding a transactivator fusion protein of the invention and a nucleic acid encoding the protein of the interest operatively linked to at least one tet operator sequence, is grown at production scale in a culture medium in the presence of tetracycline or a tetracycline analogue to stimulate transcription of the nucleotides sequence encoding the protein of interest the nucleotide sequence operatively linked to the tet operator sequence(s)) and the protein of interest is isolated from harvested host cells or from the culture medium.

Standard protein purification techniques can be used to isolate the protein of interest from the medium or from the harvested cells.

C. Production ofProteins in Vivo The invention also provides for large scale production of a protein of interest in animals, such as in transgenic farm animals. Advances in transgenic technology have made it possible to produce transgenic livestock, such as cattle, goats, pigs and sheep (reviewed in 1 Wall, R.J. et al. (1992) J. Cell. Biochem. 42:113-120; and Clark, A.J. et al. (1987) Trends in Biotechnology 1:20-24). Accordingly, transgenic livestock carrying in their genome the components of the inducible regulatory system of the invention can be constructed, wherein a gene encoding a protein of interest is operatively linked to at least one tet operator sequence.

Gene expression, and thus protein production, is induced by administering Tc (or analogue thereof) to the transgenic animal. Protein production can be targeted to a particular tissue by 20 linking the nucleic acid encoding the transactivator fusion protein to an appropriate tissuespecific regulatory element(s) which limits expression of the transactivator to certain cells.

For example, a mammary gland-specific regulatory element, such as the milk whey promoter Patent No. 4,873,316 and European Application Publication No. 264,166), can be linked to the transactivator transgene to limit expression of the transactivator to mammary 25 tissue. Thus, in the presence of Tc (or analogue), the protein of interest will be produced in the mammary tissue of the transgenic animal. The protein can be designed to be secreted into the milk of the transgenic animal, and if desired, the protein can then be isolated from the milk.

D. Animal Models of Human Disease The transcriptional activator and inhibitor proteins of the invention can be used alone or in combination to stimulate or inhibit expression of specific genes in animals to mimic the pathophysiology of human disease to thereby create animal models of human disease. For example, in a host animal, a gene of interest thought to be involved in a disease can be placed under the transcriptional control of one or more tet operator sequences by homologous recombination, as described herein). Such an animal can be mated to a second animal carrying one or more transgenes for a transactivator fusion protein and/or an inhibitor fusion protein to create progeny that carry both a tetracycline-regulated fusion protein(s) gene and a tet-regulated target sequence. Expression of the gene of interest in these progeny can be -54modulated using tetracycline (or analogue). For example, expression of the gene of interest can be downmodulated using a transcriptional inhibitor fusion protein to examine the relationship between gene expression and the disease. Such an approach may be advantageous over gene "knock out" by homologous recombination to create animal models of disease, since the tet-regulated system described herein allows for control over both the levels of expression of the gene of interest and the timing of when gene expression is downor up-regulated.

E. Production of Stable Cell Lines for Gene Cloning and Other Uses The transcriptional inhibitor systen described herein can be used keep gene expression "off' expressed) to thereby allow production of stable cell lines that otherwise may not be produced. For example, stable cell lines carrying genes that are cytotoxic to the cells can be difficult or impossible to create due to "leakiness" in the expression of the toxic genes. By repressing gene expression of such toxic genes using the 15 transcriptional inhibitor fusion proteins of the invention, stable cell lines carrying toxic genes may be created. Such stable cell lines can then be used to clone such toxic genes inducing the expression of the toxic genes under controlled conditions using Tc or analog).

General methods for expression cloning of genes, to which the transcriptional inhibitor system of the invention can be applied, are known in the art (see Edwards, C. P. and 20 Aruffo, A. (1993) Curr. Opin. Biotech. 4:558-563) Moreover, the transcriptional inhibitor system can be applied to inhibit basal expression of genes in other cells to create stable cell lines, such as in embryonic stem (ES) cells. Residual expression of certain genes introduced into ES stems may result in an inability to isolate stably transfected clones. Inhibition of transcription of such genes using the transcriptional inhibitor system described herein may be 25 useful in overcoming this problem.

Advantages The inducible regulatory system of.the invention utilizing a transactivator fusion protein addresses and overcomes many of the limitations of other inducible regulatory systems in the art. For example, very high intracellular concentrations of the transcriptional activator fusion protein of the invention are not required for efficient regulation of gene expression. Additionally, since gene expression is induced by adding rather than removing the inducing agent, the induction kinetics in the system of the invention are not limited by the rate of removal of the inducing agent and thus are typically faster. Moreover, the inducing agent is only present when gene transcription is induced, thereby avoiding the need for the continuous presence of an agent to keep gene expression off.

Use of the transcriptional inhibitor fusion proteins of the invention to inhibit transcription in eukaryotic cells also provide advantages over the use of prokaryotic repressors alone TetR, lacR) to inhibit transcription in eukaryotic cells. Since the inhibitor fusion proteins of the invention contain a eukaryotic transcriptional silencer domain, these fusion proteins should be more efficient at repressing transcription in eukaryotic cells, and thus may potentially require lower intracellular concentrations for efficient repression with less liklihood of "leakiness". Additionally, by insertion of tetO sequences into the regulatory region of an endogenous gene, the transcriptional inhibitor fusion proteins of the invention can be used to down-regulate constitutive and/or tissue-specific expression of endogenous genes.

Furthermore, in contrast to various versions of the lac system Labow et al.

(1990) Mol. Cell. Biol. 10:3343-3356; Bairn et al. (1991) Proc. Natl. Acad Sci. USA 1&:5072-5076), which are limited by the negative properties of the inducing agent (IPTG) and/or by the need to increase the temperature in order to induce gene expression (which may elicit pleiotropic effects), the inducing agent used in the system of the invention (Tc or an analogue thereof) has many advantageous properties: 1) Tc and analogues thereof exhibit 1 high affinity for TetR and low toxicity for eukaryotic cells, and thus can be used for gene 15 induction at concentrations that do not affect cell growth or morphology; 2) Tc analogues :i which retain TetR binding but which have reduced antibiotic activity exist and can be used as inducing agents, thereby avoiding possible side effects from the antibiotic property of Tc; 3) the pharmacokinetic properties ofTc and Tc analogues enable rapid and efficient cellular uptake and penetration of physiological barriers, such as the placenta or the blood-brain 20 barrier, and 4) Tc analogues with different induction capabilities permit modulation of the level of gene expression.

Thus, the invention provides an inducible regulatory system which allows for rapid activation of gene transcription without cellular toxicity and a range of induction indices.

The increase in gene expression upon induction typically is between 1000- and 2000-fold and can be as high as about 20,000-fold. Alternatively, lower levels of gene induction, fold, can be achieved depending upon which inducing agent is used. This system can be "i utilized in a wide range of applications. These applications include gene therapy, large-scale production of proteins in cultured cells or in transgenic farm animals, and the study of gene function, for example in relationship to cellular development and differentiation. Moreover, the novel transcription units of the invention allow for coordinate or independent regulation of the expression of multiple genes utilizing the regulatory components of the invention.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

-56- EXAMPLE 1: Selection of a Mutated Tet Repressor and Construction of a Tetracycline Inducible Transcriptional Activator A "reverse" Tet repressor, which binds to its target DNA in the presence rather than the absence of tetracycline, was generated by chemical mutagenesis and selection essentially as described in Hecht, B. et al. (1993) J. Bacteriology 121:1206-1210. Single-stranded DNA (coding and non-coding strands) encoding the wild-type TnlO-derived Tet repressor was chemically mutagenized with sodium nitrite. Single-stranded DNAs (40 u±g in 40 gl in Tris- EDTA buffer) were mixed with 10 Ftl of 2.5 M sodium acetate (pH 4.3) and 50 pl of sodium nitrate ranging between 0.25 M and 2 M and incubated for 45 to 60 minutes at room temperature. After mutagenesis, the complementary strand was synthesized using reverse transcriptase or by amplification using the polymerase chain reaction with Taq DNA polymerase. Since the mutagenesis procedure yields multiple mutations in the DNA, three fragments of the gene, of about 200 base pairs each, were individually subcloned into a wt Tet repressor gene in a recombinant expression vector to replace the corresponding portion of the wild-type gene. This created a pool of mutated Tet repressor genes wherein each gene had mostly single mutations in the 200 base pair mutagenized fragment of the gene.

The pool of mutated Tet repressors were screened in a genetic assay which positively selects for a functional interaction between a Tet repressor and its cognate operator using E.

20 coli. strain WH207(WH25) (the construction of this strain is described in detail in Wissmann, A. et al. (1991) Genetics 128:225-232). In this E. coli strain, tet operators direct the expression of divergently arranged p-galactoside (lacZ) and Lac repressor (lacl) genes and the lac regulatory region directs the expression of a galactokinase (galK) gene. Binding of Tet repressors to tet operators turns off transcription of the lac and lacZ genes. The 25 absence of Lac repressor allows for expression of the galK gene, which enables the E. coli strain to use galactose as a sole carbon source, which serves as one marker. The lacZphenotype serves as a second marker. Thus, bacteria containing Tet repressors which bind to tet operators have a Gal+, lacZ- phenotype. Bacteria containing wild-type Tet repressors have a Gal+, lacZ- phenotype in the absence of tetracycline. A mutated "reverse" Tet repressor (rTetR) was selected based upon a Gal+, lacZ- phenotype in the presence of tetracycline.

The nucleotide and amino acid sequence of the rTetR mutant are shown in SEQ ID NOs: 1 (nucleotide positions 1-621) and 2 (amino acid positions 1-207), respectively.

Sequence analysis of the rTetR mutant showed the following amino acid and nucleotide changes: -57an (ition affecte codon wild-typ mutant wild-type muant glu (71) lys GAA

AAA

asp (95) asn GAT

AAT

leu (101) ser TTA

TCA

gly (102) asp GGT

GAT

Two additional mutations did not result in an amino acid exchange: a psition affected codo :dt man wild-te mutant Sleu (41) leu TTG

CTG

arg (80) arg CGT CGC To convert the rTetR mutant to a transcriptional activator, a 399 base pair XbaI/Eco47mI fragment encoding amino acids 3 to 135 of rTetR encompassing the 10 mutated region) was exchanged for the corresponding restriction fragment of the expression vector pUHD15-1 to create pUHD17-1. In pUHD15-1, nucleotide sequences encoding wildtype TetR are linked in frame to nucleotide sequences encoding the C-terminal 130 amino S acids of herpes simplex virus VP 16. These transactivator sequences are flanked upstream by a CMV promoter/enhancer and downstream by an SV40 poly(A) site (the construction of 15 pUHD15-1 is described in more detail in USSN 08/076,726 and Gossen, M. and Bujard, H.

(1992) Proc. Natl. Acad. Sci. USA 19:5547-5551). Thus, in pUHDI7-1, nucleotide sequences encoding the reverse TetR mutant are linked in frame to VP16 sequences to create a reverse Tc-controlled transactivator (referred to herein as tTAR). The analogous exchange of the mutated region of rTetR for the wild-type region of TetR was performed with plasmid pUHD152-1, which is the same as pUHD15-1 except that it additionally contains nucleotide sequences encoding a nuclear localization signal linked in-frame to the 5' end of the nucleotide sequences encoding the Tet repressor. The amino acid sequence of the nuclear localization signal is MPKRPRP (SEQ ID NO: which is linked to the serine at amino acid position 2 of TetR. The resulting expression vector encoding the reverse Tc-controlled transactivator including a nuclear localization signal (referred to herein as ntTAR) was named pUHD172-1.

-58- EXAMPLE 2: Tetracycline-Induced Stimulation of Transcription by tTAR Transient Transfection The pUHD17-1 and pUHD172-1 expression vectors were transiently transfected by a standard calcium phosphate method into HeLa cells together with a reporter plasmid, pUHC13-3, in which heptameric tet operators are fused upstream of a minimal hCMV-promoter and a luciferase reporter gene (the reporter plasmid is described in detail in USSN 08/076,726 and Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad Sci. USA &:5547-5551). After incubation of the transfected cells at 37 °C for 20 hours in the presence or absence of tetracyline (or an analogue thereof), luciferase activity was assayed as follows: Cells grown to -80 confluency in 35 mm dishes in Eagle's minimal essential medium were .washed with 2 ml of phosphate-buffered saline before they were lysed in 25 mM Tris .phosphate, pH 7.8/2 mM dithiothreitol/2 mM diaminocyclohexanetetraacetic acid/10 glycerol/ Triton-X-100 for 10 minutes at room temperature. The lysate was scraped off S: 15 the culture dishes and centrifuged for 10 seconds in an Eppendorf centrifuge. Next, aliquots (10 4l) of the supernatant were mixed with 250 l1 of 25 mM glycylglycine/15 mM mM ATP and assayed for luciferase activity in a Lumat LB9501 (Berthold, Wildbad, F.R.G.) using the integral mode (10 seconds). D-Luciferin (L6882, Sigma) was used at 0.5 mM. The background signal measured in extracts of HeLa cells that did not contain a luciferase gene 20 was indistinguishable from the instrument background (80-120 relative light units sec.). Protein content of the lysate was determined according to Bradford (Bradford,

M.M.

(1976) Anal. Biochem. 72:248-254). Cells transfected with plasmids encoding either tTAR or ntTAR showed an increased level of luciferase activity in the presence of tetracyclines. This effect was consistently more pronounced when anhydrotetracycline (ATc) was used instead *25 oftetracycline.

Stable Transfection After this transient transfection analysis, expression vectors were prepared for stable transfection of cells. A pSV2neo-derived neomycin resistance cassette (described in Southern, P.J. and Berg, P. (1982) J. Mol. Appl. Genet. 1:327-341) was integrated into the transactivator expression vectors pUHD17-l and pUHD172-1, resulting in pUHD17-lneo and pUHD172-lneo, respectively. pUHD172-lneo, coding for ntTAR, was stably integrated into HeLa cells by standard techniques. Ten G418-resistant cell clones were analyzed for their phenotype by transient supertransfection with pUHC13-3 carrying the luciferase gene under the control of a minimal CMV promoter and tet operators. Three clones, HR4, and HRIO, showed a strong increase of luciferase activity in the presence of ATc. From these clones, HR5 was selected for further experiments.

To create stable transfectants for both ntTAR and a tet operator-linked luciferase reporter gene, HR5 cells were cotransfected with pUH13-3 and pHMR272, which encodes -59for hygromycin resistance (see Bernhard, H-U. et al. (1985) Exp. Cell Res. 158:237-243), and hygromycin resistant clones were selected. In an analogous experiment, HR5 cells were cotransfected with pUH13-7 and pHMR272. pUH13-7 contains a minimal promoter sequence spanning position +19 to -37 of the HSVtk promoter adjacent to the heptameric tetO sequences, rather than a minimal CMV promoter. From 21 hygromycin resistant clones, showed inducible luciferase activity upon addition of Tc or doxycyline (Dc) to the culture medium. Clones containing the luciferase reporter gene linked to a minimal CIV promoter are referred to as HR5-C, whereas those containing the luciferase reporter gene linked to a minimal tk promoter are referred to HRS-T.

Six of the HRS clones stably transfected with a ntTAR-dependent reporter unit and previously shown to be responsive to tetracyclines were grown in parallel in the absence or presence of 1 ug/ml doxycycline. About 3 x 104 cells were plated in each 35 mm dish (4 dishes for each clone). After growth for 60 hours, cells were harvested and the luciferase activity of the extracts (in relative light units (rlu)/pg extracted protein) was determined. As shown in Table 1, the absolute expression levels of six clones demonstrate that activation of luciferase gene expression over 3 orders of magnitude is achieved in several of the double stable cell lines containing the ntTAR regulatory system.

It should be noted that even higher induction factors as high as a 20,000-fold increase in expression) could be achieved if, instead of simply adding the inducing agent to 20 the culture medium, the cells were washed prior to induction and then replated in fresh culture medium containing the inducing agent.

TABLE 1. Doxycvcline-dependent luciferase activity of double stable luc+/HR5 cell clones Luciferase Activity, rlu/ig protein Clone -Doxycvcline +Doxvcvcline Induction Factor HR5-C6 65 54,911 845 :62 69,525 1120 HRS-C11 100 165,671 1660 142 179,651 1270 HRS-C14 43 44,493 1030 43 56,274 1310 HR5-T2 56 16,696 298 16,416 410 HR5-T15 6.8 1838 270 1688 260 HR5-T19 4.8 1135 236 5.4 1285 237 Induction of luciferase activity by different tetracvclines The ability of tetracycline and several different tetracycline analogues to induce luciferase expression in HR5-C11 cells was examined. HRS-C11 cells plated at a density of about 3 x 104 cells/35 mm dish confluency). After full attachment of the cells, the following tetracyclines ere added to the cultures at a concentration of 1 pg/ml: tetracycline- HCI oxytetracycline-HCl (OTc), chlorotetracycline (CTc), anhydrotetracycline-HCl (ATc) and doxycycline-HCl (Doxy). These compounds are commercially available from Sigma Chemical Co., St Louis, MO, and were kept in aqueous solution at a concentration of 1 gg/ml. Cells grown in the absence of antibiotic served as a control. After 3 days, the cells were harvested and the luciferase activity and the protein content of the extracts were determined. The results are shown in the bar graph of Figure 1. Each bar in the figure (closed and hatched) represents the relative luciferase activity (normalized toward the amount of extracted protein) of a single culture dish. The mean of the luciferase activities obtained from the two plates grown without tetracyclines was defined as 1. Tc, CTc and OTc showed modest stimulation of luciferase activity. By contrast, ATc and Doxy stimulated luciferase activity approximately 1000 and 1500 fold, respectively.

Dose-response of luciferase activity to doxvcycline in HR5-C11 cells 20 The above-described experiment examining the induction ability of different tetracyclines revealed that doxycycline was the most potent effector of the tetracyclines examined. Doxycycline was therefore selected to quantitatively analyze its dose-response.

HR5-C 11 cells were incubated with different concentrations of doxycycline and luciferase activity was measured. The data of three independent experiments are shown in Figure 2. At 25 less than 10 ng/ml in the culture medium, doxycycline is ineffective at inducing luciferase activity. However, when the concentration was raised above 10 ng/ml, an almost linear increase in expression of luciferase was observed. Maximal activation was achieved at 1 p.g/ml. At concentrations above 3 jig/ml, doxycycline showed a slight growth-inhibitory effect on HeLa cells as determined in a MTT-assay.

Kinetics of induction of the ntTAR. .yslm To examine the kinetics of doxycycline-induced ntTARmediated induction of gene expression, the time course of induction of luciferase activity in HRS-C 11 cells was monitored after addition of doxycycline to the medium (final concentration 1 ug/ml). Cells were cultured in the presence of doxycycline and after various time intervals, the cells were harvested and luciferase activity was determined as described above. As shown in Figure 3, a 100-fold induction of luciferase activity was observed after 5.5 hours incubation with Doxy.

Fully induced levels were achieved in less than 24 hours of incubation with Doxy. Thus, -61these results indicate that induction of gene expression occurs rapidly following exposure of the cells to the inducing agent.

EXAMPLE 3: Coordinate Regulation of the Expression of Two Nucleotide Sequences by a Tc-Controled Transcriptional Activator A recombinant expression vector for coordinate, bidirectional transcription of two nucleotide sequences was constructed comprising, in a 5' to 3' direction: a luciferase gene, a first minimal promoter, seven tet operator sequences, a second minimal promoter and a LacZ gene. The construct is illustrated in Figure 6. In this construct, the luciferase and LacZ genes are oriented such that they are transcribed in opposite orientations relative to the tet operator sequences, the luciferase gene is transcribed in a 5' to 3' direction from the bottom strand of DNA, whereas the LacZ gene is transcribed in a 5' to 3' direction from the top strand of DNA. The luciferase gene is followed by an SV40 polyadenylation signal, whereas the LacZ 15 gene is followed by a p-globin polyadenylation signal.

The construct was transfected into the HeLa cell line HtTA-1 cells, which express a wild-type Tet repressor-VP 16 fusion protein (referred to as tTA and described in Gossen, M.

and Bujard, H. (1992) Proc. Natl. Acad Sci. USA 82:5547-5551). The tTA fusion protein binds to tet operator sequences in the absence of Tc (or analogue) but not in the presence of 20 Tc (or analogue). The construct was cotransfected into HtTA-1 cells with a plasmid which confers hygromycin resistance and stably transfected clones were selected based upon their hygromycin resistant phenotype. Selected hygromycin resistant (Hygr clones were .examined for luciferase and p-galactosidase activity. Clones positive for all three markers (Hygrr, luc+, P-gal+) were then examined for tetracycline-dependent coregulation of 25 expression of luciferase and p-galactosidase activity by cultureing the clones in increasing amounts of tetracycline and measuring luciferase and p-galactosidase activity. The results of such an experiment using clone Htl316-8/50 are shown in Figure 8. In the absence of tetracycline (in which case tTA can bind-to tet operators and activate gene expression), both luciferase and p-galactosidase activity is detected. In the presence of increasing amounts of tetracycline, luciferase and p-galactosidase activity are coordinately and equivalently downregulated. This data demonstrates that expression of two genes can be coordinately regulated by a tetracycline-controlled transactivator by operatively linking the two genes to the same tet operator sequence(s).

EXAMPLE 4: Construction of a Tetracycline-Regulated Transcriptional Inhibitor Fusion Protein Comprising TetR and a Krueppel Silencer Domain To contruct an expression vector encoding a tetracycline-regulated transcriptional inhibitor of the invention (also referred to as a tetacycline controlled silencer domain, or -62tSD), a nucleic acid fragment encoding a transcriptional silencer domain is ligated into an expression vector containing nucleotide sequences encoding a wild-type or modified mutated) TetR such that the silencer domain coding sequences are ligated in-frame with the TetR coding sequences. The plasmid pUHDl41sma-l contains nucleotide sequences encoding a wild-type Tnl0-derived Tet repressor (the nucleotide and amino acid sequences of which are shown in SEQ ID NOs: 16 and 17, respectively). In pUHD141sma-l, the TetR coding sequence is linked at its 5' end to a CMV promoter and at its immediate 3' end to a nucleotide sequence that creates a polylinker into which additional nucleic acid fragments can be introduced. The nucleotide sequence across this polylinker region is: TCC CCG GGT AAC TAA GTA AGG ATC C (SEQ ID NO: 24) (wherein TCC CCG GGT ACC encode amino acid residues 205-208 of TetR, namely Ser-Gly-Ser-Asn). This polylinker region includes restriction endonuclease sites for PspAI (CCC GGG) and BamHI (GGA TCC).

Downstream of the polylinker region, the plasmid contains an SV40-derived polyadenylation "signal. The pUHD141sma-l vector is illustrated schematically in Figure 11.

15 To construct an expression vector encoding a fusion protein between TetR and a transcriptional silencer domain from the Drosophila Krueppel (Kr) protein, a nucleic acid fragment encoding a silencer domain from Kr is amplified by the polymerase chain reaction (PCR) using Kr cDNA as a template. Oligonucleotide primers are designed which amplify a nucleic acid fragment encoding the C-terminal 64 amino acids of Kr (referred to as C64KR).

This region corresponds to amino acid positions 403-466 of the native protein. The nucleotide and amino acid sequences of C64KR are shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively. PCR primers are designed to include restriction endonuclease sites such that the resultant amplified fragment contains restriction endonuclease sites at its 5' and 3' ends. Restriction endonuclease sites are chosen that are contained within the polylinker of 25 pUHD141sma-l which allow in-frame, directional ligation of the amplified fragment into the polylinker site. For example, PCR primers are designed which incorporate a PspAI site (CCC GGG) at the 5' end of the fragment encoding C64KR and a BamHI site at the 3' end of the fragment. After a standard PCR reaction, the amplified fragment and pUHD 41-smal are digested with PspAI and BamHI. The amplified fragment is then ligated directionally into the polylinker site of pUHDl41-smal using standard ligation conditions to create the expression vector pUHD141kr-l. Standard techniques are used to isolate the desired plasmid and confirm its construction. Construction ofpUHD141kr-1 is illustrated schematically in Figure 11.

The resultant pUHD141kr-l expression vector contains nucleotide sequences encoding a fusion protein comprising amino acids 1-207 of the wild type TetR linked inframe to amino acids 403-466 ofKr (C64KR). The nucleotide and amino acid sequences across the junction of the fusion protein are as follows: AGT GGG TCC CCG GGT GAC ATG GAA (SEQ ID NO: 25) and Ser-Gly-Ser-Pro-Gly-Asp-Met-Glu (SEQ ID NO: 26). Ser- -63- Gly-Ser corresponds to amino acids 205-207 of TetR, Pro-Gly are encoded by the polylinker and Asp-Met-Glu correspond to the amino acids 403-405 of C64KR.

Similarly, an expression vector encoding a fusion protein of a mutated TetR (that binds to tetO only in the presence of Tc) and C64KR can be constructed as described above using nucleotide sequences encoding a mutant TetR (the nucleotide and amino acid sequences of which are shown in SEQ ID NOs: 18 and 19, respectively) in place of the wild type TetR sequences in pUHD141-smal.

An expression vector encoding a TetR-Kr fusion protein pUHD141kr-l) is transiently or stably transfected into host cells as described in Example 2 to express the TetR- Kr fusion protein in the host cell. A reporter gene construct containing one or more tetO sequences, a minimal promoter and a reporter gene, such as luciferase, is also transfected into the cells as described in Example 2. (Reporter gene constructs are described in further detail in USSN 08/076,726 and Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 8:5547-5551). Luciferase activity in the presence and absence of increasing concentrations 15 of Tc or an analogue, doxycycline, is measured as described in Example 2. For the wild type TetR-Kr fusion protein described above, the transcriptional inhibiting ability of the fusion protein is determined by comparing the amount of luciferase activity in the presence of "doxycycline (no repression) to the amount of luciferase activity in the absence of doxycycline (repression). The transcriptional inhibiting activity of the fusion protein can also be tested using reporter gene constructs that exhibit higher basal levels of expression higher levels of expression in the presence of doxycycline) by using a reporter gene construct that contains S additional positive regulatory elements enhancer sequences).

EXAMPLE 5: Construction of a Tetracycline-Regulated Transcriptional Inhibitor 25 Fusion Protein Comprising TetR and a v-erbA Silencer Domain To construct an expression vector encoding a fusion protein between TetR and a transcriptional silencer domain from the v-erbA oncogene product, a nucleic acid fragment encoding a silencer domain from v-erbA is ligated in-frame into pUHD141sma-l as described in Example 4. A nucleic acid fragment encoding a v-erbA silencer domain suitable for ligation into pUHD141sma-l is amplified by the polymerase chain reaction (PCR) using a v-erbA cDNA as a template. Oligonucleotide primers are designed which amplify a nucleic acid fragment encoding amino acids 364-635 of the native v-erbA protein. The nucleotide and amino acid sequences of this region of v-erbA are shown in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. As described in Example 4, PCR primers are designed such that the amplified v-erbA fragment contains restriction endonuclease sites at its 5' and 3' ends, such as PspAI at the 5' end and BamHI at the 3' end. After a standard PCR reaction, the amplified fragment and pUHD141-smal are digested with PspAI and BamHI. The amplified fragment is then ligated directionally into the polylinker site of pUHD141-smal using standard ligation -64conditions to create the expression vector pUHDl41kr-l. Standard techniques can be used to isolate the desired plasmid and confirm its construction. Construction ofpUHD141erb-1 is illustrated schematically in Figure 11.

The resultant pUHD141erb-1 expression vector contains nucleotide sequences encoding a fusion protein comprising a wild type TetR linked in-frame to amino acids 364- 635 of v-erbA. The nucleotide and amino acid sequences across the junction of the fusion protein are as follows: AGT GGG TCC CCG GGT CTG GAC GAC (SEQ ID NO: 27) and Ser-Gly-Ser-Pro-Gly-Leu-A-AsAsp (SEQ ID NO: 28). Ser-Gly-Ser corresponds to amino acids 205-207 of TetR, Pro-Gly are encoded by the polylinker and Leu-Asp-Asp correspond to amino acids 364-366 of the v-erbA silencer domain.

As described in Example 4, an expression vector encoding a fusion protein of a mutated TetR (that binds to tetO only in the presence of Tc) and a v-erbA silencer domain can be constructed as described above using nucleotide sequences encoding a mutant TetR (the nucleotide and amino acid sequences of which are shown in SEQ ID NOs: 18 and 19, 15 respectively) in place of the wild type TetR sequences in pUHD141-smal.

Expression of the TetR-v-erbA fusion protein in host cells and assaying of the transcriptional inhibiting activity of the fusion protein is as described in Example 4 for the TetR-Kr fusion protein.

EXAMPLE 6: Regulation of Gene Expression in Transgenic Animals by tTAR To examine the ability of tTAR to regulate gene expression in vivo, transgenic strains of mice were constructed which contained heterologous chromosomal insertions of either a tTAR expression construct or a reporter gene operably linked to tet operators. Single transgenic strains containing either a tTAR expression construct or the tetO-linked reporter gene were then cross bred and double transgenic progeny were identified. The double transgenic animals were then characterized as to the ability of tTAR, in a tetracycline dependent manner, to regulate expression of the reporter gene. This example demonstrates that tTAR effectively stimulates the expression of a gene operably linked to tet operators in tissues of the animals in vivo upon administration of tetracycline (or analogue) to the animals, whereas expression of the tetO-linked gene remains at background levels in the absence of tetracycline or an analogue. These results demonstrate that the tetracycline-controlled transcriptional regulatory system described herein functions effectively in animals, in addition to cell lines in vitro.

Generation of mice transenic for a P V-tTAR ssi on Mice expressing tTA protein were obtained by pronuclear injection into fertilized oocytes of a 2.7kb XhoI-Pfml fragment excised from plasmid pUHG17-1. This DNA fragment contained the tTAR gene (shown in SEQ ID NO: 1) under the transcriptional control of the human CMV IE promoter (position +75 to -675) together with a rabbit P-globin polyadenylation site including an intron. The human CMV IE promoter is a constitutive promoter that allows expression of the mutated tetR-VP 16 fusion protein in all cells where chromosomal integration of the DNA sequence encoding tTAR has occurred. DNA was injected into fertilized oocytes at a concentration of approximately 5 ng per 1 by standard techniques. Transgenic mice were generated from the injected fertilized oocytes according to standard procedures. Transgenic founder mice were analyzed using polymerase chain reaction (PCR) and Southern hybridization to detect the presence of the tTAR transgene in chromosomal DNA of the mice. Two transgenic mouse lines, CR3 and CR4 were identified and crossbred with another transgenic mouse line carrying a luciferase reporter gene under the control of tetO sequences. (described further below).

:Generation of mice transgenic for the PhCMV* luciferase reporter unit Mice carrying a PhCMV*- luc reporter gene expression unit were generated by 15 pronuclear injection into fertilized oocytes of a 3.1 kb Xhol-Eael fragment excised from plasmid pUHC13-3. This DNA-fragment contains the luciferase gene under transcriptional control of the tetracycline-responsive PhCMV*-I promoter (SEQ ID NO: together with an SV40 t early polyadenylation site including an intron. DNA was injected into oocytes at a concentration of approximately 5 ng per pl and transgenic mice were generated according to standard procedures. Transgenic founder mice were analyzed using Southern hybridization to detect the presence of the PhCMV*-1 luc transgene in chromosomal DNA of the mice. A mouse line transgenic for the tetO-linked luciferase reporter gene, L7, was crossbred with the tTAR transgenic lines CR3 and CR4 (described further below).

Generation of mice translenic for the PhCMV-jluc and PhCM tAR Having constructed single transgenic mice expressing tTAR or carrying PhCMV*luc, double transgenic mice carrying both the tTA expression vector and the luciferase reporter-units were obtained through cross breeding ofheterozygous mice transgenic for one of the two transgenes. Double transgenic animals were identified by standard screenings PCR and/or Southern hybridization) to detect the presence of both the tTAR transgene and the PhCMV*- luc transgene in chromosomal DNA of the mice.

Induction and analysis of luciferase activity in tissue samples from mice For oral administration, tetracycline or its derivative doxycycline were given in the drinking water at a concentration of 200 gg per ml with 5 sucrose to hide the bitter taste of the antibiotics. For lactating mice, the concentration was 2 mg per ml with 10 sucrose to ensure a sufficient uptake via the milk by the young.

To analyze luciferase activity, mice were killed by cervical dislocation and tissue samples were homogenized in 2 ml tubes containing 500 gl lysis-buffer (25 mM Tris -66phosphate, pH 7.8/2 mM DTT/ 2 mM EDTA/ 10 glycerol/I Triton X100) using a Ultra-Turrax. The homogenate was frozen in liquid nitrogen and centrifuged after thawing for 5 min at 15,000g. 2-20 pl of the supernatant were mixed with 250 tI luciferase assay buffer (25 mM glycylglycine, pH 7.5/15 mM MgSO4/ 5 mM ATP) and luciferase activity was measured for 10 sec after the injection of 100Il of a 125 liM luciferin solution using Berthold Lumat LB 9501. The protein concentration of the homogenate was determined using Bradford assay and luciferase activity was calculated as relative light units (rlu) per pg of total protein.

Results Mice from 2 lines carrying the PhCMV-tTAR transgene (CR3 and CR4) were mated *with mice from line L7, transgenic for PhCMV*-1 luc. The L7 line shows a very low but detectable background of luciferase activity in different organs that is probably due to position effects at the integration side. The background luciferase activity in different tissues 15 of the L7 single transgenic mice is illustrated graphically in Figure 12, represented by the "checked columns on the right-hand side for each tissue examined (each column represents the results from one animals). The luciferase activity in different tissues of the C3/L7 double -transgenic mice in the absence of the tetracycline analogue doxycycline uninduced conditions) is illustrated graphically in Figure 12, represented by the dark columns in the middle for each tissue examined. The luciferase activity in different tissues of the C3/L7 double transgenic mice in the presence of doxycycline induced conditions) is illustrated graphically in Figure 12, represented by the light columns on the left-hand side for each tissue examined.

Luciferase activity was detectable in the seven tissues of the double transgenic mice examined: pancreas, kidney, stomach, muscle, thymus, heart and tongue. The tissue pattern of activated luciferase levels in the presence of doxycycline) in the double transgenic mice was similar to expression patterns of the hCMV IE promoter reported in the literature.

This is consistent with expression of the luciferase reporter gene being regulated by tTAR (which is expressed in the mice under the control of the hCMV IE promoter). The level of reporter gene induction varied among the different tissues examined. Regulation factors up to 100,000 fold 5 orders of magnitude) were achieved, e.g. in the pancreas.

FQL1T.ALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

67 SEQU CE LISTING GENERAL INFORMATION: i) APPLICANTS: NAME: Hermann Bujard STREET: Remler Str. 9 CITY: D-69120 Heidelberg COUNTRY: Germany NAME: Manfred Gossen STREET: 978 Arlington Boulevard #B CITY: El Cerrito STATE: California CE) COUNTRY: USA POSTAL CODE (ZIP): 94530 (ii) TITLE OF I!.ENTION: Tetracycline-Regulatef-- Modulators 20 (iii) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE

ADDRESS:

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NAME: DeConti, Giulio A. Jr.

REGISTRATION NUMBER: 31,503 -68- REFERENCE/DOCKET NUOMBER: BBI-009C2PC TELECOMMUNICATION

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TELEPHONE: (617)227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO:1: r r r r r r r r r r SEQUENCE CHARACTERISTICS: LENGTH: 1008 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: exon LOCATION: 1..1008 (ix) FEATURE: NAME/KEY: mRNA LOCATION: 1..1008 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

misc. binding 1..207 misc. binding 208..335 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1005 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG TCT AGA TTA GAT AAA AGT AAA GTG ATT AAC AGC GCA TTA GAG CTG Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 CTT AAT GAG GTC GGA ATC- GAA GGT TTA ACA ACC Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr CGT AAA CTC GCC CAG Arg Lys Leu Ala Gln AAG CTA GGT GTA Lys Leu Gly Val GAG CAG CCT ACA CTG TAT TGG CAT GTA AAA AAT AAG Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 96 144 192 CGG GCT TTG CTC GAC GCC TTA GCC ATT GAG ATG TTA GAT AGO CAC CAT Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His ACT CAC TTT TGC CC Thr His Phe Cys P1 S AAT AAG GCT AAA AC Asn Lys Ala Lys S4 GCA AAA GTA CAT T( Ala Lys Val His S4 100 CTC GAA AAT CAA T Leu Giu Asn Gin L 115 AAT GCA TTA TAT G Asn Ala Leu Tyr A 130 GTA TTG GAA GAT C Val Leu Giu Asp G 145 CCT ACT ACT GAT Pro Thr Thr Asp S 3 TTT GAT CAC CAA C 30 Phe Asp His Gin C 180 ATC ATA TGC GGA Ile Ile Cys Gly 195 TAC AGC CGC GCG Tyr Ser Arg Ala 210 CTG CTC GAT CTC Leu Leu Asp Leu 225 GCT CCG CGC CTG Ala Pro Arg Leu ACG GCC CCC CCG Thr Ala Pro Pro 260 GGC GAG GAC GTG Gly Giu Asp Val 275 CTG GAC ATG TTG Leu Asp Met Leu 290 .r TTA A; :o Leu L) 70 ;T TTT AC Br Phe A3 CA GAT A er Asp TI TA GCC T eu Ala P: CA CTC A .a Leu S 1 AA GAG C in Giu H 150 rGT ATG C er Met P .65 ;GT GCA C ;ly Ala C rir GAA Leu Glu CGT ACG Arg Thr CCG GAC Pro Asp 230 TCC TTT Ser Phe 245 ACC CAT Thr Asp GCG ATG Ala Met GGG CAC Gly Asp

IAGGG

r' Gly

;ATGT

:g Cys A CGG ir Arg rT TTA he Leu 120 GC GCT or Ala 35 AT CAA is Gin :CG CCA 'ro Pro ;AG CCA iu Pro kAA CA; ys Glr 20C AAA AA( Lys Asr 215 GAC GAI Asp As] CTC CC Leu Pr GTC AG Val Se GCG CA Ala Hi 28 GGG G; Gly A 295 -69- GAA AGC TGG CA Glu Ser Trp Gi 75 GCT TTA CTA AG Ala Lou Lou Se 90 CCT ACA GAA A; Pro Thr Giu L) 105 TGC CAA CAA G( Cys Gin Gin G: GTG GGG CAT T Val Giy His P1 1.

GTC GCT AAA G Val Ala Lys G 155 TTA TTA CGA C Leu Leu Arg G 170 GCC TTC'TTA 1 Ala Phe Leu P 185 CTT AAA TGT C 1 Lou Lys Cys C AAT TAC GGG *i Ann Tyr Gly GCC CCC GAA p Ala Pro Glu 235 C GCG GGA CAC o Ala Gly His 250 C CTG GGG GAC r Lou Giy Asp 265 r GCC GAC GCG .s Ala Asp Ala I0 T TCC CCG GGT ip Ser Pro Gly A GAT n Asp IT CAT !r His A AG rs Gin ;T TTT Ly Phe 125 rT ACT he Thr AA GAA iu Glu AA GCT in Ala TC GGC 'he Gi AA AGI iu Se2 LCT AC oer Thz 220 GAG GC 3iu Al ACG CG Thr Ar GAG CT Glu Le CTA G; Leu As 28 CCG G( Pro G 300 irT TTA CGC Phe Lou Arg so CGC AAT GGA Arg Ann Gly TAT GAA ACT Tyr Giu Thr 110 TCA CTA GAG Ser Leu Glu TTA GGT TGC Leu Gly Cys AGG GAA ACA Arg Giu Thr 160 ATC GAA TTA Ile Giu Leu 175 CTT GAA TTG Leu Giu Leu 190 GGG TCC GCG Gly Ser Ala C ATC GAG GGC c Ile Giu Gly 3 GGG CTG GCG a Gly Lou Ala 240 C AGA CTG TCG g Arg Lou Ser 255 C CAC TTA CAC u His Lou Asp 270 IC CAT TTC CAT ip Asp Pho Asp ;A TT ACC CCC Ly Phe Thr Pro 240 289 336 384 432 480 528 576 624 672 720 768 816 864 912 CAC GAC TCC 0CC CCC TAC 0CC OCT CTO OAT ATO GCC QAC TrC GAG TTT His Asp Ser Ala Pro Tyr Gly Ala Lou Asp Met Ala Asp Phe Giu Phe 305 310 315 320 GAG CAG ATG TT ACC GAT CCC CTT OGA ATT GAC GAG TAC GOT 000 TAG Glu Gin Met Phe Thr Asp Pro Leu Gly Ile Asp Giu Tyr Gly Gly 325 330 335 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 335 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Giu Lei 1 5 10 is Leu Asn Giu Val Giy Ile Giu Gly Leu Thr Thr Arg Lys Leu Ala Gi 25 30 Lys Leu Gly Val Giu Gin Pro Thr Leu Tyr Trp His Val Lys Asn Ly 40 45 25 Arg Ala Leu Leu Asp Ala Leu Ala Ile Giu Met Leu Asp Arg His Hi so 55 Thr His Phe Cys Pro Leu Lys Giy Giu Ser Trp Gin Asp Phe Leu Ar 65 70 75 Asn Lys Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asn Gl es 90 Ala Lys Val His Ser Asp Thr Arg Pro Thr Glu Lys Gln Tyr Giu TI 100 105 110 Leu Giu Asn Gin Leu Ala Phe Leu Cys Gin Gin Giy Phe Ser Leu G 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Giy C 130 135 140 Val Leu Giu Asp Gin Giu His Gin Val Ala Lys Giu Giu Arg Giu T 145 150 155 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gin Ala Ile Giu 1 165 170 175 Phe Asp His Gin Gly Ala Giu Pro Ala Phe Leu Phe Giy Leu Giu I 180, 185 190 lie Ile Cys Gly Leu Giu Lys Gln Leu Lys Cys Giu Ser Gly Ser 195 200 205 960 1009 a n

S

99 0 Lu ys hr ,eu eu kla Tyr Ser Arg 210 Leu Leu Asp 225 Ala Pro Arg Thr Ala Pro 260 Gly Glu Asp 275 Leu Asp Met 290 His Asp Ser 20 305 Glu Gin Met Ala Leu Leu Pro Val Leu Ala Phe Arg Pro Ser 245 Thr Ala Gly Pro Thr 325 Thr Asp 230 Phe Asp Met Asp Tyr 310 Asp Lys Asn Asn 215 Asp Asp Ala Leu Pro Ala Val Ser Leu 265 Ala His Ala 280 Gly Asp Se 295 Gly Ala Lei Pro Leu Gl -71- Tyr Gly Ser Thr 220 Pro Glu Glu Ala 235 SGly His Thr Arg 250 Gly Asp Glu Leu SAsp Ala Leu Asp 285 r Pro Gly Pro Gly 300 u Asp Met Ala Asp 315 y Ile Asp Glu Tyr 330 Ile Glu i Gly Leu Arg Leu 255 His Leu 270 Asp Phe Phe Thr Phe Glu Gly Gly 335 Gly Ala 240 Ser Asp Asp Pro Phe 320 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAC GCG CTA GAC GAT TTC GAT CTG GAC ATG TTG Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 1 5 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal -72- (xi) SEQMENCE DESCRIPTZON. SEQ ID 11:4: Asp Ala Lau ASP Asp Pb. ASP Lou Asp Me" Lou 1 5 5(2) INFOPM.TION FOR SEQ XD 110:5: Wi SEQUECE CgARACTENSTICS: Lm=GT: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FAGET TYPE:. internal (xi) SEQUENCE DESCRIPTION: SEQ ID 110:5: 20 Met Pro Lys Arg Pro Arg Pro INFORMATION FOR SEQ ID NO:6: SE.QuEIcE CHARACTERISTICS: LENGTH: 569 base pairs TYPE: nucleic acid sTR.ANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA be. 35 (xi) SEQuENCE DESCRIPTION: SEQ ID 110:6: GAATTCGGGG CCGCGGAGGC TGGATCGGTC CCrGGTGTCTT CTATGGAGGT CAAAACPIGCG 6 TGGATGGCGT CTCCAGGCGA TCTGACGGTT cAcTAAAcGA GCTCTGCTTA TATAGGTCGA 120 GTTTACCACT CCCTATCAGT GATP.QAGAAA AGTGAAAGTC GAGTTTACCA CTCCCTATCA 2.80 GTGATAGAOA AAAGTGAAAG TCGAGTTTAC CACTCCCTAT CAGTGATAGA GAAAAGTCAA 240 AGTCGAGTTT AccAcTCCCT ACCAGTGATA GAGAAAAGTrG AAAGTCGAGT TTACCACTCC 300 -CTATCAGTGA TAGAGAAAAG. TGAAAGTCGA GTTTACCACr CCCTATCAGT GATAGAGAAA 360 AGTGAAAGTC GAGrTTACCA CTCCCA GTGATAGAGA AAAGTGAAAG TCGAGCTCGG, 420 TACCCGGGTC GAGTGGCG1 GTACGGTGGG AGGCCTATAT AAG~P.GAGCT CGTTTAGTGA 480 ACCGTCAGAT CGCCTGGAGA cGCCATCCAC GCTGTTTTGA CCTCCATAr-A AG-ACACCGGG 540 ACCGATCCAG CCTCCGCGGC CCCGAA=T 569 NFop,4ATION FOR SEQ ID 11:7: -73- Wi SROCCE ~~TZ

CS:

LOE: 520 bagge Pairs' TYPE:. nucleic acid SIeDUsS: double CD) TOPOLOGY: linear (ii) MOLECE TYPE: DNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AGATCTGCArs GGTCGCTCGG TCGA= C ACACGCGTC ACUTTAATAT GCGAAGTGGA ccGGAT~CTCGAGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAArGT CGAGTTTACC 120 ACTCCC1ATC AGTGATArzAG AAAAGTGAAA GTCGAGT1"TA CCACTCCCTA TCAGTGATAG AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 240 TAccAcTc CCTATCAGTG ATAGAGAAAA GTGAAAGTCG AGTTYACCAC TCCCTATCAG 300 *TGATAGAGAA AAGTGAAAGT CrmGLTTACC ACTCCCTATC AGTGATAGAG AAAAGTGAAA 360 GTCGAGCTCG; GTACCCGGGT CGAGTAGGCG TGTACG=TG GAGGCTATA TAAGCAGAGC 420 TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG 480 AAGACACCGG GACCGATCCA GCCTCCGCGG CCCCGAATTC 520 INFORMATION FOR SEQ ID NO: 8: Ci) SEQUENCE CELARACTERISTICS: LENGT: 450 base pairs CB) TYPE: nucleic acid STRA4DzEDNES~s: double TOPOLO0GY: linear (ii) MOLECULE TYPE: DNA (genoUtic) Cvi) ORIGIN4AL SOURCE: ORGANISM: Humani cytomegalovirus STRAIN: X12, Townle (ix) FEATURE: NAME/KEY: MRNA LOCATION: 382. .450 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: GAATTCCTCG AG=ZACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTI TACC AcTCCCTATC AGTrATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCATGA.TAG 120 AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGCAG 180 TTcCACTc CCTATcAGTG ATACGAGAAAA GTGAAAGTCG AGTTTACCAC TCCCTATCAG 240 -74- TO=ATAA AAQTGAAAGT CGA=TITACC ACTCCL-nC AMMUPM AA""AAA, GTCMW.CTCr. GTACCCGGGT CGAGTAGGCG TGTACG=WG GAGGCCTAT1 TAAGCAGAGC TCGTTTA=T AP.CCGTCAGA TCGCCTGGAG ACG=VTCCA CGCTGTTTTG A~CCCATAG AAGACACCGG GACCGATCCA GCCTCCG CG S a

OSS*

a.

0 0

S.

S

*000 *0S* eaSe

S

*0 S

S

*5 a a.

a.

a a.

*5

S

a INFORMATION FOR SRQ ID 110:9: SEQUENCE CHARACTERISTICS: LENGTH: 450 base Pairs JB) TYPE: nucleic acid STILINDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Human cytomegaloviriB STRAIN: Townie (ix) FEATURE: NAME/XEY mRNA LOCATION: 392. .450 Cxi) SEQUENCE DESCRIPTION: SEQ ID 110:9: GAATTCCTCG ACCCGGGTAC CGAGCTCGAC TTTCACTTTT CTCTATCACT 25 GGTAAACTCG ACTT~cAcTT TTCTCTATCA cTG-ATAGGGA GTGGTAAACT TrTrCTCTAT CACTGATAGG GAGTGGTAAA CTCGACTrrC AcTTTTCTCT GGGAGTOGTA AACTCGACTT TCACTTTTCT CTATCACTGA TP.OGGAGTGG TTCAcTTT CTCTATCACT GATAGGGAGT GGTAAACTCG AcrrTCTT CTGATAGGGA GTGGTAAACT CGAGTAGGCG TGTACGGTGG GAGGCC'ATA TCGTrTAGTG AACCGTCAGA TCGCCTGGAG AGcrxaaccA CGCTGTTTTG AAGACACCGG GACCGATCCA GCCTCCGCGG

GATAGGGAGT

CGACTTTCAC

ATCACTGATA

TAAACTCGAC

TTCTCTATCA

TAAGCAGAGC

ACCTCCATAG

120 IS0 240 300 360 420 450 INFORMATION FOR SEQ ID 110:10: SEQUENCE CHARACTERISTICS: LENGTH: 398 base Pairs TypE: nucleic acid STRAmEDNESS: double CD) TOPOLOGY: lineoar (ii) MOLECULE TYPE: DN (genoinic) (vi) ORIG31L SOURCE: ORG1IISN: Herpes Simplex Vfr~m STRAIN: 108 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAGCTCGACT TTCACTTTTC TCTATCACTG ATAGGGAGTG GTAA CCGA XXTCACTTT TCTVTATCAC TGATAGGGAG TGGTrAAACTC GACTTTCACT TTTCTATC ACTGATAGGG12 AGTGGTAAAC TCGACTTTCA, CTTTTCTCTA TCACTGATAG GGA.GTrGTAAL ACTCGACTIT ISO CACTTTTCTC TATCACTGAT AGGGAGTGGT AA CTCGACT TTCACTTTTC TCTATCACTG 240 ATAGGGAGTG GTAAACTCGA. CTTTCACTTT -TCTCTATCAC TGATAGGGAG TGGTAAACTC 300 GAGATCCGGc GAATTCGAAc AcGCAmaTGC AGTcGGGGCG GCGCGGTCCG AGGTCCAcTT 360 CGCATATTAA GGTG.ACGcGT GTGGCCTCGA ACACCGAG 398 INFORM4ATION FOR SEQ ID NO:l1: Wi SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNSS: double TOPOLOGY: linear (ii) MOLECUL TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID No:11: ACTTTATCAC TGATAAACAA ACTATCAGT GATAAAGA 38 25 INFORMTION FOR SEQ ID NO:12: i)SEQUENCE CHuACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid sTRANDEDNEss: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO :12: ACTCTATCAT TGATAGAGTT CCCTATCAGT GATAGAGA 38 InaORmaTIO FOR SEQ iD 310:13:- -76- Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 38 base pairs TYPE: nucleic acid CC) STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGCTTATCAT CGATAAGCTA GTTTATCACA GTTAAATT INFORMATION FOR SEQ ID NO:14: Wi SEQUENCE CHARACTERISTICS: LENGTH: 38 ba 'se pairs tmj ixkE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ACTCTATCAT TGATAGGGAA, CTCTATCAAT GATAGGGA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA xi) SEQUENCE DESCRIPTION: SEQ ID AATCTATCAC TaATAGAGTA cccT.ATCATC GATAGAGA INFORMATION FOR SEQ-ID NO:16: Wi SEQUENCE CHARACTERISTICS: CA) LENGTH: 621 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: -77- ATG TCT AGA TTA GAT AAA AGT AAA MTG ATT AAC AGC GCA TTA GAG CTG Met Ser Arg Lou Asp Lys Ser Lys Val Ile 1

CTT

Lou

AAG

LYS

CG

Arg

ACT

Thr

AAT

Asn

GCA

Ala

CTC

30 Leu

AAT

Asn 35

GTA

Val 145

CCT

Pro

TTT

Phe

ATC

Ile

AAT

Asn

CTA

Lou

GCT

Ala so

CAC

His

AAG

Lys

AAA

Lys

GAA

Giu

GCA

Ala 130

TTG

Leu

ACT

Thr

OAT

Asp

ATA

Ile

GAG

Glu

GGT

Gly

TTG

Leu ?1-r Phe

GCT

Ala

GTA

Val

AAT

Asn 115 Leu

GAA

Glu

ACT

Thr

CAC

His

TGC

Cys 195

GTC

Val

GTA

Val

CTC

Leu

TGC

Cys

AAA

Lys

CAT

His 100

CAA

Gin

TAT

Tyr

GAT

Asp

GAT

Asp

CAA

Gin 180

GGA

Gly 5

GGA

Gly

GAG

Giu

GAC

Asp

CCT

Pro

AGT

Ser

TTA

Leu

TTA

Leu

GCA

Al a

CAA

Gin

AGT

Ser 165

GGT

Gly

TTA

Leu

PATC

Ile

CAG

Gin

GCC

Ala Leu 70

TTT

Phe

GGT

Gly

GCC

Ala

CTC

Leu

GAG

Giu 150

ATG

Met

GCA

Ala

GAA

Glu GAA GOT TTA Giu Gly Lou 25 CCT ACA TTG Pro Thx Lou 40 TTA GCC A?? Leu Ala Ile 55 GAA G00 GAA Giu Giy Giu AGA TGT OCT Arg Cys Ala ACA COG CCT Thr Arg Pro 105 TTT TTA TGC Phe Leu Cys 120 AGC GCT GTO Ser Ala Val 135 CAT CAA GTC His Gin Val CCG CCA TTA Pro Pro Leu GAG CCA GCC Giu Pro Ala 185 AAA CAA CTT Lys Gin Lou 10

ACA

Tmx

TAT

Tyr

GAG

Giu

AGC

Ser

TTA

Leu 90

ACA

Thr

CAA

Gin Giy

GC'I

Ala

TT.

Leu 170C Phe Asn~

ACC

Thr

TGG'

Trp

ATO

Met

TG

Trp 75

CTA

Leu

GAA

Giu

CAA

Gin

CAT

His

'AAA

Lys 155

CGA

Arg Leu

:GT

A.rg

CAT

Hlis

TTA

Leu

CAA

Gin

AGT

Ser

AAA

Lys

GT

Gly

TTT

Phe 140

GAA

Oiu

CAA

Gin

TTC

Phe

AAA

Lys

GTA

Vai

OAT

Asp

GAT

Asp

CAT

His

CAG

Gin rrr Phe 125

ACT

Thr

GAA

Giu

OCT

Ala

GGC

Gly

CTC

Lou

AAA

Lys

AGO

Arg Phe

CGC

Arg

TAT

Tyr 110

TCA

Ser Leu

AGO

Arg

ATC

Ile

C??

Leu 190 GCC CAG Ala Gin AAT AAG Aen Lys CAC CAT His His

COT

Leu Arg OAT GGA Asp Oly GAA ACT Giu Thr CTA GAO Leu 0Th GOT TOC Cly Cys GAA ACA Oiu Thr 160 GAA TTA Giu Leu 175 GAA ??G 0Th Leu Ser Ala Lou Glu Lou 48 96 144 192 240 288 336 384 432 480 528 576 621 AAA TOT Lys Cys GAA AGT 000 TCC Glu Ser Oly Ser 205 INFORMATION FOR SEQ ID NO:i7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 207 amino acids TYPE: amino acid TOPOLOGY: linear 78 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:17: Met 5cr Arg Lau Asp 1 Lys Ser Lys Val Ile Ann Ser Ala Leu. Glu Leu.

Lau Lys Arg Thr An Ala Lou An Val 145 Pro Phe Ile kan Glu Val Gly Ile Giu Gly Leu, Ala so His Lys Lys Glu Ala 130 Leu Thr Asp I114 Gly 35 Lou Phe Ala Val Asn 115 Leu Glu Thr His ~Cys 195 Val Glu Leu Asp Cys Pro Lys Ser His Leu 100 Gin Leu Tyr Ala Asp Gin Asp Ser 165 Gin Gly 180 Gly Lou Gln Ala Lou 70 Phe Gly Ala Leu Giu 150 Met Pro Lou 55 Giu Arg Thr Phe Ser 135 His Pro ThrJ 40 Ala Gly Cys Arg Leu 120 Ala Gin Pro Leu 25 Leu Ile Giu Ala Pro 105 Cys Val Val Leiu Thr Tyr Glu Ser Leu 90 Thr Gin Gly Ala Leu 170 Phe r rp Met Trp 75 Leu Giu Gin His Lys 155 Arg LeL ArgJ His Leu Gin Ser Lys Gly Phe 140 Glu Gin Phe Liys VIal Asp Asp His Gin Phe 125 Thr Glu Ala Gly Leu Lys Arg Phe Arg Tyr 110 Ser Lou Arg Ile Lou k.sn His Lou Asp Glu Lou Gly Giu Glu 175 Giu Gin Lys His Arg s0 Gly Thr Giu Cys Thr 160 Lou Leu Ala Giu Pro Ala 185 190 Giu Lys Gin Lou Lys Cys Glu Ser Gly Ser 200 205 INFORMATION FOR SEQ ID NO:iS: Wi SEQUENCE CHARACTERISTICS: LENGTH: 621 base pairs TypE: nucleic acid STRANDEDNESS: double TOPOL~OGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 79- ATM TCT AGA WA MAT AAA A=T AAA GMG ATC AAc A=C =C TTA GAG Cmo Met Ser Arg Lou Asp Lys Ser Lys Val Ile

S.

CTT

Lou.

AAG

Lys

CGG

Arg

ACT

Thr

AAT

An

GCA

Ala

CTC

30 Leu

AAT

An 35

GTA

Val 145

CCT

Pro

TTT

Phe

ATC

Ile

AAT

Asn

CTA

Leu

GCT

Ala so

CAC

His

AAG

Lys

AAA

Lys

GAA

Giu

GCA

Al a 130

TTG

Leu

ACT

Thr

CAT

Asp

ATA

Ile

GAG

Glu

GGT

Gly

TG

Leu

TIT

Phe

GCT

Ala

GTA

Val

AAT

An 115

TTA

Leu

GAA

Giu

ACT

Thr

CAC

His

TGC

Cys 195

GTC

Val

GTA

Val

CTC

Leu

TGC

Cys

AAA

Lys

CAT

His 100

CAA

Gin

TAT

Tyr

GAT

Asp

GAT

Asp

CAA

Gin 180

GGA

Gly

GGA

Gly

GAG

Glu

GAC

Asp

CCT

Pro

AGT

Ser

TCA

Ser

TTA

Leu

GCA

Ala

CAA

Gin

AGT

Ser 165

GGT

Gly

TTA

Lou

ATC

Ile

CAG

Gin

CC

Ala

TA

Leu 70 Trr Phe

CAT

Asp

GCC

Ala

CTC

Leu

GAG

Giu 150

ATG

Met

GCA

Ala

GAA

Giu GAA4 Giu

CCT

Pro

TTA

Leu.

55

AAA

Lys.

AGA

Arg

ACA

Thr Trr Phe

AC

Ser 135

CAT

His

CCG

Pro

GAG

Glu

AAA

Lys 2iy

A.CA

Thr

GCC

Ala

GGG

Gly

TGT

Cys

CCC

Arg

TTA

Leu 120

GCT

Ala

CAA

Gin

CCA

Pro

CCA

Pro

CAA

Gin 200 Lou Leu

ATT

Ile

CAA

Glu

GCT

Ala

CCT

Pro 10s

TGC

Cys

GTG

Val

GTC

Val

TTA

Leu

GCC

Ala 185 10

ACA

Thr

TAT

Tyr

GAG

Glu

AGC

Ser

TTA

Leu 90

ACA

Thr

CAA

Gin

CG

Gly

GCT

Ala T TA Lou 170

TTC

Phe An

ACC

Thr

TG

Trp

ATG

Met

TGG

Trp 75

CTA

Leu

GAA

Giu

CAA

Gin

CAT

His

AAA

Lys 155

CGA

Arg

TTA

Leu Ser

CGT

Arg

CAT

His

TTA

Leu

CAA

Gin

ACT

Ser

AAA

Lys

GGT

Gly

TTT

Phe 140

GAA

Clu

CAA

Gin Phe Lau

CTC

Lou.

AAA

Lys

AGG

Arg Phe

CGC

Arg

TAT

Tyr 110

TCA

Ser

TTA

Leu

AGO

Arg

ATC

Ile

CTT

Leu Giu is

CC

Ala

AAT

An

CAC

His

TTA

Leu

AAT

An

GAA

Glu

CTA

Leu

GOT

Gly

GAA

Giu

GAA

Glu 175

GAA

Glu Lou.

CAG

Gin

AAG

Lys

CAT

His

CC

Arg

GCA

Gly

ACT

Thr

GAG

Giu

TGC

Cys

ACA

Thr 160

TTA

Leu

TTG

Leu 48 96 144 192 240 288 336 384 432 480 528 576 CTT AAA TCT GAA AGT COG TCC Leu Lys Cys Giu Ser Cly Ser 205 INFORMATION FOR SEQ ID NO:19: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 207 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ Met Ser Arg Lou Asp Lys Ser Lys Val Lau Lys Arg Thr Asn Ala Leu Asn 30 Vai 145 Pro Phe Ile Asn Leu Ala so His Lys Lys Giu Ala 130 Leu Thr Asp Ile Glu Gly 35 Lou Phe Ala Val Asn 115 Lou Giu Thr His Cys 195 Val Val Leu Cys Lys His 100 Gin Asp Asp Gin 180 Gly Gly Giu Asp Pro Ser as Ser Leu Ala Gin Ser 165 Gly Leu Ile Glu Gin Pro Ala Leu 55 Leu Lys 70 Phe Arg Asp Thr Ala Phe Leu Ser 135 Giu His 150 Met Pro Ala Giu Giu Lys Gly Thr 40 Ala Gly Cys Arg Lou 120 Ala Gin Pro Pro Leu 25 Leu Ile Giu Ala Pro 105 Cys Val Val Lou Ala 185 ID NO: 19: le Asn Ser Thr Thr Arg Tyr Trp His Giu Met Leu Sor Trp Gin 75 Leu Leu Ser 90 Thr Giu Lys Gin Gin Gly Gly His Phe 140 Ala Lys Giu 155 Lou Arg Gin 170 Phe Leu Phe Ala Lys Val Asp Asp His Gin Phe 125 Thr Giu Ala Gly Lou Lou Lys Arg Phe Arg Tyr 110 Sor Lou Arg Ile Lou 190 Giu is Ala Asn His Leu Asn Giu Lou Gly Giu Glu 175 Glu Lou Gin

LYS

His Arg Gly Thr Giu Cys Thr 160 Leu Leu Gin Lou Lys Cys Giu Ser Gly Ser INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 192 base pairs TYPE: nucleic acid STRAZ4DEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID 81 GAC ATG GAA AAA GCG ACA CCG GAG ACG ATG GTC CAT TGG ATT TGT CTG 48 Asp Met Glu Lys Ala Thr Pro Giu Thr met Val His Trp Ile Cys Leu, 1 5 10 AAG ATG GAG CCA. OCT CTG TGG ATC; GCC ATT ACA GCA ACA TCG CAC GOC 96 Lys Met Glu Pro Ala Leu Trp Met Ala le Thx Ala Thr Ser His Gly 25 GCA AGG CAC AGG ACA TTC GTC 000 TTT TCC GGC TGC CTC CAC CGC AAA 144 Ala Arg His Arg Thr Phe Val Gly Phe Ser Gly Cys Leu His Arg Lys 40 TCC CTC ACG TAC CCA GTG ATA TGC CTG AGC AAA CCG AGC CAG AGG ATT 192 Ser Leu Thr Tyr Pro Val Ile Cys Leu Ser Lys Pro Ser Gin Arg le so 55 INFORMATION FOR SEQ ID 110:21: 20 SEQUENCE CHARACTERISTICS: LENGTH: 64 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:21: Asp Met Glu Lys Ala Thr Pro Giu Thr Met Val His Trp Ile Cys Leu *1 5 10 Lys Met Glu Pro Ala Leu Trp Met Ala Ile Thr Ala Thz Ser His Gly 25 Ala Arg His Arg Thr Phe Val Giy Phe Ser Gly Cys Leu His Arg Lys 40 .Ser Leu Thr Tyr Pro Val Ile Cys Leu Ser Lys Pro 5cr Gin Arg Ile 55 INFORMATION FOR SEQ ID 110:22: SEQUENCE CHARACTERISTICS: LENGTH: 816 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID 110:22: CTG GAC GAC TCG AAG CGC GTA GCC AAG COG AAG CTG ATC GAG G.AG AAC 48 Lau Asp Asp Ser Lys Arg Val Ala Lys Arg Lys Lou Ile Glu Glu An 1 5 10 1s -S2 CGG GAG CGG CGA MGC AAG GAG GIAG ATG ATC AAA Arg Giu Arg Arg Arg Lys Giu Glu ccc Pro

GCG

Ala

AAA

Lys

GAC

Asp

ATC

Ile

ATG

Met

TGC

Cys

GAG

35 Giu 145

CAG

Gin 40 CrC Leu

CTG

Leu

TGC

Cys

GAG

Gilu 225

AGC

Ser

CAC

His so TrC Phe

GG

Gly

ACG

Thr TrC Phe

TGC

Cys 130

AGC

Ser

TTG

Leu

GGC

Gly

CTG

Leu

GTG

Val 210

CAC

His

CCC

Pro CaC Arg

CTG

Leu

GAC

Asp

CCG

Pro

TCG

Ser 115

ATG

Met

GAA

Glu

AAG

Lys

AAG

Lys

GAG

Gin 195

GAT

Asp

TAC

Tyr

AGC

Ser

AGC

Ser

CTC

Leu

AAA

Lys

GCC

Ala 100

GAG

Giu

GAG

Giu

ACG

Thr

AAC

An

TCG

Ser 180

GCC

Ala

AAG

Lys

ATC

Ile GCA GAG Ala Giu ACC AAC Thr An GAA GAT Giu Asp 70 GTG GAC Val Asp a5 ATC ACC Ile Thr CTG CCG Leu Pro ATC ATG le Met CTG ACG Leu Thr 150 GGA GGG Gly Giy 165 CTG TCT Leu Ser GTG CTG Val Leu ATA GAG Ile Giu AAC TAC Ann Tyr 230

GAG

Glu

GCG

Ala 55

ATC

Ile

CTG

Leu

CGC

Arg

TGC

Cys

TCG

Ser 135

CTG

Leu

CTG

Leu

GCC

Ala

CTC

Leu

AAG

.Lys 215

CGC

Arg

TGG

Trp 40

CAG

Gin

GGT

Gly

GAG

Giu

GTG

Val

GAG

Glu 120

CTG

Leu

AGC

Ser

GGG

Gly TrC Phe

ATG

Met 200

TGC

Cys

AAA

Lys Met Ile Lys 2S GAG CTG ATC Giu Leu Ile GGC AGC CAC Gly Ser His CAG TCG CCC Gin Ser Pro 75 GCG TTC AGC Ala Phe Ser 90 GTC GAC TTT Val Asp Phe 105 GAT GAG ATC Asp Gin Ile CGC GCC GCC Arg Ala Ala GGG GAA ATG Giy Giu Met 155 GTC GTG TCT Val Val Ser 170 AAC CTG GAC Asn Leu Asp 185 TCC TGA GAC Ser Ser Asp GAG GAG TCG Gin Glu Ser CAC AAC ATT His Ann Ile 23S

TCC

Ser

CAC

His

TGG

Trp

ATG

Met

GAG

Giu

GCC

Ala

ATC

le

GTG

Val 140

GCC

Ala

GAT

Asp

GAC

Asp

CGG

Arg

TAC

Tyr 220 ccc Pro

CTG

Leu

GTG

Vai

AAG

Lys

GCC

Ala

TTT

Phe

AAA

Lys

CTG

Leu 125

CGC

Arg

GTC

Val

GCC

Ala

ACC

Thr

ACG

Thr 205

CTG

Leu

CAC

His

CAG

Gin

GTG.

Val

CAG,

Gin

TCC

Ser

ACA

Thr

AAC

Asn 110

CTG

Leu

TAC

Tyr

AAA

Lys

ATC

Ile

GAG

Giu 190

GGG

Gly

CTG

Leu

TTC

Phe

CAC

His

ACC

Thr

AGG

Arg

ATG

Met

AAA

Lys

CTG

Leu

AAG

Lys

GAC

Asp

CGC

Arg

TTC

Phe 175

GTG

Vai

CTG

Leu

GCG

Ala

TGG

Trp

CGG

Arg

GAG

Giu

AGG

Arg

CTT

Leu

ATC

le ccc Pro

GGC

Gly

CCC

Pro

GAG

Giu 160

GAC

Asp

GCC

Ala

ATC

Ile

TTC

Phe

TCC

Ser 240 96 144 192 240 288 336 384 432 480 528 576 624 672 720 83 AAG CTG CTG ATG AAG GTG GCM GAC CTG COC ATG ATC GGC GCC TAC CAC Lys Loeu Lau Met Lys Val Ala Asp Lau Arg Met Ile Gly Ala Tyr His 245 250 255 GCC AGC CGC TYC amG CAC ATG AAm GTm G= TOC ccc ACC GG cTc TCC Ala Ser Arg Pile Lou His Met Lye Val Giu Cys Pro Thr Glu Lou 8cr 260 265 270 INFORMATION FOR SEQ ID N0:23: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 272 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 768 816 a Lou Arg Pro Ala Lys 65 Asp Ile Met Cys Giu 145 Gin Leu (xi) SEQUENCE Asp Asp Ser Lys 5 Glu Arg Arg Arg Ser Pro Ser Ala 3S His Arg Ser Thr 50 Phe Lou. Lou Giu Gly Asp Lys Val Thr Pro Ala Ile 100 Pile Ser Giu Lou 115 Cys Met Glu Ile 130 Ser Glu Thr Lou Lou Lys Asn Gly 165 Giy Lys Ser Lou 190 Arg Lys Giu Asn Asp 70 Asp Thr Pro Met Thr 150 Gly Ser Val Glu Glu Ala 55 Ile Lou Arg Cys Ser 135 Leu Leu Ala Ala Giu Trp 40 Gin Gly Giu Val Giu 120 Lou Ser Gly Pile Lys Met 25 Glu Gly Gin Ala Val 105 Asp Arg Gly Val Asn 185 Arg 10 Ile Lou Ser Sor Pho 90 Asp Gin Ala Giu Val 170 Leu Lys Lys Ile His Pro 75 Ser Pile Ile Ala Met 155 Ser Asp Leu Ser His Trp Met Giu Ala Ile Val 140 Ala Asp Asp Ile Lou Val Lys Ala Phe Lys Lou 125 Arg Val Ala Thr Glu Glm Vai Gin Ser Thr Asn 110 Lou Tyr Lys Ile Giu 190 DESCRIPTION: SEQ ID NO:23: G1u is His Thr Arg Met Lys Lou Lys Asp Arg Pile 175 Val Asn Arg Giu Arg Leu Ile Pro Gly Pro Glu 160 Asp Ala Lau Lou Gin Ala Val Leu Lou Met Ser Ser Asp Arg Thr Gly Lou Ile 195 200 205 -34- Cys Val Asp Lys Ile 210 Giu His Tyr Ile An Giu Lys 215 Cys Gin Glu 8cr 22S Lys Leu. Lou Met Lys Tyr 230 Val His Arg Lys His Asn Ile 235 Ala Asp Lou Arg Met 250 Met Lys Vai Giu Cys 26S Tyr Lou Lou Ala Ph.

220 Pro Rio Ph. Trp 8cr 240 Ile Gly Ala Tyr His 255 Pro Thx Giu Lou Ser 270 Ala Ser Arg Phe Lou 260

C

INFORMATION FOR SEQ ID NO:24: Wi SEQUENCE CHARACTERISTICS: LENGTH; 25 base pairs TYPE: nucleic acid CC) STRANDEDNESS: double TOPOLOGY: linear 20 (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TCCCCGGGTA ACTAAGTAAG GATCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs CE) TYPE: nucleic acid sTRANDEDNEss: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Cxi) SEQUENCE DESCRIPTION: SEQ ID 35 AGTGGGTCCC CGGGTGACAT GGAA INFORMATION FOR SEQ ID NO:26: Ci SEQUENCE CHARACTERISTICS: LENGTH: a amino acids TYPE: amino acid CD) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 8cr Gly 5cr Pro Gly ASP Met GlU 1 INFORMATION FOR SEQ ID NO:27: Wi SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRhNDED1,TBS: double.

TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AGTGGGTCCC CGGGTCTGGA CGAC 24 INFORMATION FOR SEQ ID NO:28: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 8 aminoacd TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Ser Gly Ser Pro Gly Leu Asp Asp *1

Claims

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.

2. The nucleic acid of claim 1, wherein the first polypeptide is a mutated Tet repressor.

3. The nucleic acid of claim 2, wherein the mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor.

4. The nucleic acid of claim 2, wherein the mutated Tet repressor has at least one amino acid addition or deletion compared to a wild-type Tet repressor.

5. The nucleic acid of claim 3, wherein the mutated Tet repressor is a mutated 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.

6. The nucleic acid of claim 5, wherein the mutated Tnl0-derived Tet repressor comprises the amino acid sequence corresponding to positions 1 to 207 of SEQ ID NO: 2.

7. The nucleic acid of claim 1, wherein the second polypeptide comprises a transcription activation domain of herpes simplex virion protein 16.

8. The nucleic acid of claim 7, wherein the transcription activation domain comprises a C-terminal amino acid region of herpes simplex virus virion protein 16 comprising an amino acid sequence corresponding to positions 208 to 335 of SEQ ID NO: 2.

9. The nucleic acid of claim 7, wherein the transcription activation domain comprises at least one copy of a C-terminal region of herpes simplex virus virion protein 16 comprising an amino acid sequence shown in SEQ ID NO: 4. The nucleic acid of claim 1, wherein the second polypeptide comprises a transcription activation domain selected from the group consisiting of: an acidic transcription activation domain, a proline-rich transcription activation domain, a serine/threonine-rich transcription activation domain and a glutamine-rich transcription activation domain. -87-

11. The nucleic acid molecule of any one of claims 1 to 10, wherein the fusion protein further comprises an operatively linked third polypeptide which promotes transport of the fusion protein to a cell nucleus.

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.

13. The fusion protein of claim 12, wherein the first polypeptide is a mutated Tet repressor.

14. The fusion protein of claim 12 or 13, wherein the second polypeptide comprises a transcription activation domain of herpes simplex virion protein 16. %1 5. The fusion protein of claim 12, which comprises an amino acid sequence shown in SEQ ID NO:2. ~16. A recombinant vector comprising the nucleic acid molecule of claim 1 in a form suitable for expression of the fusion protein in a host cell. •17. The recombinant vector of claim 16, wherein expression of the fusion protein is regulated by at least one tissue-specific regulatory element.

18. The recombinant vector of claim 16, wherein expression of the fusion protein is •regulated by at least one tet operator sequence.

19. A host cell comprising the recombinant vector of claim 16. The host cell of claim 19, further comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

21. The host cell of claim 20, wherein the nucleotide sequence to be transcribed is an exogenous nucleotide sequence introduced into the host cell. -88-

22. The host cell of claim 20, wherein the nucleotide sequence to be transcribed is an endogenous nucleotide sequence to which at least one tet operator sequence has been operatively linked.

23. The host cell of claim 20, which is a mammalian cell.

24. The host cell of claim 21, which is a human cell. The host cell of claim 20, which is a yeast, insect or fungal cell.

26. A method for stimulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the host cell of claim 20 comprising contacting the host cell with tetracycline or a tetracycline analogue.

27. The method of claim 26, wherein the tetracycline analogue is anhydrotetracycline, doxycycline or cyanotetracycline.

28. The method of claims 26 or 27, further comprising isolating a protein encoded by the nucleotide sequence operatively linked to the at least one tet operator sequence from .the host cell or from a culture medium in which the host cell is grown.

29. A non-human transgenic organism comprising a transgene comprising the nucleic acid molecule of claim 1 in a form suitable for expression of the fusion protein in cells of the non-human transgenic organism.

30. The non-human transgenic organism of claim 29, wherein the transgene is integrated at a predetermined location within the genome of the organism.

31. The non-human transgenic organism of claim 29, further comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

32. The non-human transgenic organism of claim 30, further comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

33. A method for stimulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the non-human transgenic organism of 88a- claim 31, comprising administering tetracycline or a tetracycline analogue to the organism.

34. The method of claim 33, wherein the tetracycline analogue is anhydrotetracycline, doxycycline or cyanotetracycline.

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 V V* *o -89- operator sequence operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.

36. The nucleic acid of claim 35, wherein the first polypeptide binds to a tet operator sequence in the absence but not the presence of tetracycline or a tetracycline analogue.

37. The nucleic acid of claim 36, wherein the first polypeptide is a Tet repressor.

38. The nucleic acid of claim 37, wherein the first polypeptide comprises an amino acid sequence shown in SEQ ID NO: 17.

39. The nucleic acid of claim 35, wherein the first polypeptide binds to a tet operator :sequence in the presence but not the absence of tetracycline or a tetracycline analogue.

40. The nucleic acid of claim 39, wherein the first polypeptide is a mutated Tet repressor.

41. The nucleic acid of claim 40, wherein the mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor.

42. The nucleic acid of claim 40, wherein the mutated Tet repressor has at least one "amino acid addition or deletion compared to a wild-type Tet repressor.

43. The nucleic acid of claim 41, wherein the mutated Tet repressor has an amino acid substitution at at least one amino acid position corresponding to an amino acid position 25 selected from the group consisting of position 71, position 95, position 101 and position 102 of a wild-type TnlO-derived Tet repressor amino acid sequence.

44. The nucleic acid of claim 43, wherein the mutated Tet repressor comprises an amino acid sequence shown in SEQ ID NO: 19. The nucleic acid of claim 35, wherein the second polypeptide comprises a transcription silencer domain of a v-erbA oncogene product

46. The nucleic acid of claim 45, wherein the second polypeptide comprises an amino acid sequence shown in SEQ ID NO: 23.

47. The nucleic acid of claim 35, wherein the second polypeptide comprises a transcription silencer domain of a Drosophila Krueppel protein.

48. The nucleic acid of claim 47, wherein the second polypeptide comprises an amino acid sequence shown in SEQ ID NO: 21.

49. The nucleic acid of claim 35, wherein the second polypeptide comprises a transcription silencer domain of a protein selected from the group consisiting of the retinoic acid receptor alpha, the thyroid hormone receptor alpha, the yeast Ssn6/Tupl protein complex, the Drosophila protein even-skipped, SIRI, NePI, the Drosophila dorsal protein, TSF3, SFI, the Drosophila hunchback protein, the Drosophila knirps protein, WTI, Oct-2.1, the Drosophila engrailed protein, E4BP4 and The nucleic acid molecule of claim 35, wherein the fusion protein further comprises an operatively linked third polypeptide which promotes transport of the fusion protein to a cell nucleus. 15 51. A fusion protein which inhibits transcription in eukaryotic cells, comprising a first polypeptide which binds to a ret operator sequence operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells.

52. The fusion protein of claim 51, wherein the first polypeptide binds to a tet operator 20 sequence in the absence but not the presence of tetracycline or a tetracycline analogue.

53. The fusion protein of claim 52, wherein the first polypeptide comprises an amino acid S. sequence shown in SEQ ID NO: 17.

54. The fusion protein of claim 51, wherein the first polypeptide binds to a tet operator sequence in the presence but not the absence of tetracycline or a tetracycline analogue. The fusion protein of claim 54, wherein the first polypeptide comprises an amino acid sequence shown in SEQ ID NO: 19.

56. The fusion protein of claim 51, wherein the second polypeptide comprises a transcription silencer domain of a v-erbA oncogene product.

57. The fusion protein of claim 56, wherein the second polypeptide comprises an amino acid sequence shown in SEQ ID NO: 23.

58. The fusion protein of claim 51, wherein the second polypeptide comprises a transcription silencer domain of a Drosophila Krueppel protein. -91-

59. The fusion protein of claim 58, wherein the second polypeptide comprises an amino acid sequence shown in SEQ ID NO:21. The fusion protein of claim 51, wherein the second polypeptide comprises a transcription silencer domain of a protein selected from the group consisting of the retinoic acid receptor alpha, the thyroid hormone receptor alpha, the yeast Ssn6/Tupl protein complex, the Drosophila protein even-skipped, SIR1, NeP the Drosophila dorsal protein, TSF3, SFI, the Drosophila hunchback protein, the Drosophila knirps protein, WT1, Oct-2.1, the Drosophila engrailed protein, E4BP4 and

61. A recombinant vector comprising the nucleic acid molecule of claim 35 in a form suitable for expression of the fusion protein in a host cell.

62. A host cell comprising the recombinant vector of claim 61. The host cell of claim 62, further comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

64. The host cell of claim 63, wherein the nucleotide sequence to be transcribed is an exogenous nucleotide sequence introduced into the host cell.

65. The host cell of claim 63, wherein the nucleotide sequence to be transcribed is an *C endogenous nucleotide sequence to which at least one tet operator sequence has been operatively linked.

66. A method for modulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the host cell of claim 63, comprising modulating the concentration of tetracycline or a tetracycline analogue that is in contact with the host cell.

67. A non-human transgenic organism comprising a transgene comprising the nucleic RA acid molecule of claim 35 in a form suitable for expression of the fusion protein in cells the non-human transgenic organism. -92-

68. The non-human transgenic organism of claim 67, wherein the transgene is integrated at a predetermined location within the genome of the organism.

69. The non-human transgenic organism of claim 67, further comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

70. The non-human transgenic organism of claim 68, further comprising a nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence.

71. A method for modulating transcription of the nucleotide sequence operatively linked to the at least one tet operator sequence in the non-human transgenic organism of claim 69, comprising modulating the concentration of tetracycline or a tetracycline analogue present in the organism.

72. The method of claim 71, wherein the tetracycline analogue is anhydrotetracycline, doxycycline or cyanotetracycline.

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. 1 P 74. The host cell of claim 73, wherein the first polypeptide binds to a tet operator sequence in the presence, but not the absence of tetracycline or a tetracycline analogue 92a and the third polypeptide binds to a tet operator sequence in the absence, but not the presence of tetracycline or tetracycline analogue. The host cell of claim 73, wherein the first polypeptide binds to a tet operator sequence in the absence, but not the presence of tetracycline or a tetracycline analogue and the third polypeptide binds to a tet operator sequence in the presence, but not the absence, of tetracycline or tetracycline analogue.

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. a. a a -93- a second transen encoding a second fusion protein which inhibits transcription, the second fusion protein comprsing 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.

77. The non-human transgenic animal of claim 76, wherein the first polypeptide binds to a tet operator sequence in the presence, but not the absence of tetracycline or a tetracycline analogue and the third polypeptide binds to a tet operator sequence in the absence, but not the presence of tetracycline or a tetracycline analogue.

78. The non-human transgenic animal of claim 76, wherein the first polypeptide binds to a tet operator sequence in the absence, but not the presence of tetracycline or a tetracycline 15 analogue and the third polypeptide binds to a tet operator sequence in the presence, but not the absence, of tetracycline or a tetracycline analogue. S: 79. A recombinant vector for coordinate, bidirectional transcription of a first and a second nucleotide sequence to be transcribed, the vector comprising 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 25 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. The recombinant vector of claim 79, wherein the first and second nucleotide sequence to be transcribed have been introduced into the first and second cloning sites, respectively.

81. The recombinant vector of claim 80, wherein the first and second nucleotide sequences encodes polypeptide subunits of a heterodimeric protein.

82. The recombinant vector of claim 80, wherein the first nucleotide sequence encodes a protein of interest and the second nucleotide sequence encodes a detectable marker. -94-

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.

84. The composition of matter of claim 83,.wherein either the first or the second class type of tet operator sequence is a class B tet operator, sequence. The composition of matter of claim 83, wherein the first and second nucleotide sequence to be transcribed have been introduced into the first and second cloning site, respectively.

86. The composition of matter of claim 85, wherein the first nucleotide sequence comprises a therapeutic gene and the second nucleotide sequence comprises a suicide gene.

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 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 25 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. *o

88. The kit of claim 87, further comprising a third container means containing a third 30 nucleic acid 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.

89. The kit of claim 88, further comprising a fourth container means containing a tetracycline or a tetracycline analogue. 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, substantially as herein described with reference to one or more of the examples and accompanying drawings.

91. 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, substantially as herein described with reference to any or more of the examples and accompanying drawings.

92. A recombinant vector comprising a nucleic acid encoding a fusion protein which activates transcription the fusion protein comprising a first polypeptide which binds to a i 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, substantially as herein described with reference to one or more of the examples and accompanying drawings. .o 93. A host cell comprising a recombinant vector incorporating a 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, substantially as herein described with reference to any or more of the examples and accompanying drawings. -96-

94. A method for stimulating transcription of a nucleotide sequence operatively linked to the at least one tet operator sequence in a host cell comprising contacting the host cell with tetracycline or a tetracycline analogue, substantially as herein described with reference to one or more of the examples and accompanying drawings.

95. A non-human transgenic organism with cells incorporating a transgene comprising a 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, substantially as herein described with reference to one or more of the examples and accompanying drawings.

96. An isolated nucleic acid encoding a fusion protein which inhibits transcription, the fusion protein comprising a polypeptide which binds to a tet operator sequence in the absence but not presence of tetracycline or a tetracycline analogue, substantially as herein described with reference to one or more of the examples and accompanying drawings.

97. A fusion protein which inhibits transcription in eukaryotic cells, the fusion protein comprising a polypeptide which binds to a tet operator sequence and which is operatively linked to a heterologous polypeptide which inhibits transcription in eukaryotic cells, •substantially as herein described with reference to one or more of the examples and accompanying drawings.

98. A method for modulating transcription of a nucleotide sequence operatively linked to the at least one tet operator sequence in a host cell comprising modulating the concentration of tetracycline or a tetracyclic analogue that is in contact with the host cell, -97- substantially as herein described with reference to one or more of the examples and accompanying drawings.

99. A recombinant vector for coordinate, bidirectional transcription of a first and second nucleotide sequence to be transcribed, and incorporating at least one tet operator sequence, and wherein transcription of the first and second nucleotide sequences proceeds in opposite directions relative to the at lest one tet operator sequence, substantially as herein described with reference to one or more of the examples and accompanying drawings.

100. 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, that 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; substantially as hereinbefore described with reference to one or more of the examples a •o .and accompanying drawings.

101. A kit comprising a carrier means having in close confinement therein at least two container means of a first and a second nucleotide sequence and incorporating at least one tet operator sequence, the first container means containing a first nucleic acid encoding a fusion protein which activates transcription and comprises a polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline c q4,nanalogue and which is operatively linked to a second polypeptide which activates -98- transcription in eukaryotic cells, and the second container means containing a second nucleic acid comprising a cloning site operatively linked to at least one tet operator sequence, substantially as herein before described with reference to one or more of the examples and accompanying drawings. DATED this 14th Day of January 2002 BASF AKTIENGESELLSCHAFT AND KNOLL AKTIENGESELLSCHAFT Attorney: DAVID A. ADAMTHWAITE Fellow Institute of Patent and Trade Mark Attorneys of Australia of BALDWIN SHELSTON WATERS S g ooo o o *o o*° *o *o* a* oo o *o oo *ooo o