WO1998038322A1

WO1998038322A1 - Nucleic acid constructs for durable transgene expression

Info

Publication number: WO1998038322A1
Application number: PCT/EP1998/000992
Authority: WO
Inventors: Hendrik Veelken; Albrecht Lindemann
Original assignee: KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
Priority date: 1997-02-25
Filing date: 1998-02-20
Publication date: 1998-09-03
Also published as: DE19707493C1; AU6622798A

Abstract

Nucleic acid constructs suitable for expressing a foreign gene in a host cell have the following components: (a) at least part of a promotor which corresponds to a host-related, constitutive active promotor; (b) at least one binding site for a transcription factor; (c) at least one transgene; and (d) at least one gene coding for a transcription factor that can bond to the binding site for a transcription factor (b).

Description

Nucleic acid constructs for long term expression of

Transgenes

The nucleic acid constructs according to the invention are used for the expression of transgenes in host cells. Genetic engineering makes it possible to transfer certain genes from a donor into recipient cells. This transferred gene (transgene) is then expressed in the host cell. In higher organisms (eukaryotes) in particular, regulation of expression involves considerable difficulties. The present invention relates to nucleic acid constructs, in particular vectors, which enable permanent, long-term expression of transgenes in transformed cells.

Hofmann et al. (Proc. Natl. Acad. Sci. USA, Vol. 93 (May 1996), pp. 5185-5190) describe an autoregulatory cassette which enables the reversible induction of transgene expression as a function of tetracycline. The Hofmann et al. The nucleic acid construct described uses a minimal promoter derived from the cytomegalovirus as the promoter. In its natural form, this promoter controls the expression of the very early proteins of cytomegalovirus (CMV Immediate Early Minimal Promoter). The expression of foreign genes in eukaryotic cells is often controlled by viral promoters, since the so-called "long terminal repeat elements" of retroviruses, or the "immediate / early promoter" of the cytomegalovirus have proven to be very effective promoters.

However, it has been found that viral promoters do not allow long-term expression of transgenes, since these promoters are "switched off" relatively quickly by the host cell. This can possibly be done by hypermethylation of the promoter region.

It is therefore an object of the present invention to provide a nucleic acid construct which enables long-term, sufficient expression of transgenes in eukaryotic cells, which in a preferred embodiment can be regulated.

The present invention therefore relates to nucleic acid constructs which are suitable for expressing a foreign gene in a host cell and which have the following components:

(a) at least part of a promoter which corresponds to a host-related, constitutively active promoter,

(b) at least one binding site for a transcription factor,

(c) at least one transgene and

(d) at least one gene coding for a transcription factor which can bind to the binding site for a transcription factor (b). The term “nucleic acid construct” is understood to mean an arrangement of various components which consist of nucleic acid, in particular DNA. The components of the nucleic acid constructs according to the invention act functionally on one another and enable the expression of a transgene under controlled conditions. The individual components of the nucleic acid constructs according to the invention can preferably be located on one nucleic acid construct or they can be distributed over two or more constructs. However, the prerequisite is that in the latter case the individual nucleic acid constructs are in one cell.

The nucleic acid constructs according to the invention are used to express a foreign gene in a host cell. For the purposes of the present invention, the term “foreign gene” or “transgene” is understood to mean a gene which codes for a gene product which, after its expression, causes certain effects. In the context of the present invention, the transgenes are preferably genes which code for cytokines. In the context of the present invention, the term cytokines encompasses a group of immunomodulatory proteins, which are also called immunotransmitters. The cytokines act as humoral regulators that regulate the functional activities of certain cells. The term "cytokines" includes the interleukins, interferons and colony stimulating factors. In the context of the present invention, the colony-stimulating factors, in particular G-CSF (granolocyte colony-stimulating factor), are particularly preferred.

In the nucleic acid constructs according to the invention, expression is increased by incorporating at least one binding site for a transcription factor at a suitable location in the promoter. The gene coding for the transcription factor is arranged downstream of the promoter and is controlled by it. The The binding site for the transcription factor, which is incorporated into the promoter according to the invention, preferably originates from a different gene arrangement and therefore does not occur in the corresponding naturally occurring promoter.

The nucleic acid constructs according to the invention contain, as a special feature within a constitutively active promoter, a binding sequence for a transcription factor and the gene of the same transcription factor at the 3 'end of the construct. In a preferred embodiment of these constructs, the translation of the gene of this transcription factor is made possible by suitable fusion with the "internal ribosome entry sequence" of the encephalomyocarditis virus. In such a construct, the expression of the transgene and the transcription factor is controlled by the same promoter. Such a construct is therefore also called "dicistronic". By incorporating the binding sequence for the transcription factor into the promoter, the expression of the transcription factor has a feedback-enhancing effect on its own expression and on the expression of the transgene in the arrangement according to the invention. According to the invention, the constitutively active function of the promoter is significantly enhanced by this mechanism.

In a preferred embodiment, the nucleic acid constructs according to the invention can be regulated in terms of expression. Such nucleic acid constructs have the following components:

(a) at least part of a promoter that corresponds to a host-related promoter,

(b) at least one binding site for a repressor,

(c) at least one transgene and (d) a chimeric gene coding for a repressor, which can bind to the binding site for a repressor (b), fuses with at least part of a transactivation factor.

A binding site for a repressor is then coupled to the functional part of the promoter. This binding site can preferably be located within the promoter. Furthermore, the nucleic acid construct contains a chimeric gene that codes for a repressor that can bind to the binding site for the repressor. The chimeric gene also contains a gene for a transactivation factor. When the substrate for which the repressor is specific is present, the chimeric gene product binds to the substrate, thereby preventing binding to the repressor binding site. However, if the substrate is not present in the cell, the repressor binds to the binding site for the repressor. The increase in expression is then brought about by the other part of the chimeric gene product, namely the transactivation factor.

In the context of the present invention, a preferred regulation system is the tetracycline repressor system, which originates from the transposon 10 from E. coli.

In this case, the regulatory substrate is tetracycline. If tetracycline is present in the cell, the repressor and tetracycline bind and the chimeric gene product cannot bind to the tet operator. This does not increase transcription. If there is no tetracycline in the cell, the product of the chimeric gene can bind to the tet operator and an increase in transcription, which leads to an increase in expression, takes place via the VP16 protein of the herpes simplex virus.

In a preferred embodiment of the present invention, the binding site for the repressor (b) is tet operator, which is present several times in a tandem-like arrangement in a particularly preferred embodiment.

In another preferred embodiment, an internal ribosome entry sequence (IRES) is arranged between the transgene (c) and the gene coding for the transcription factor or the chimeric gene (d), the IRES of the encephalomyocarditis virus being particularly preferred.

In a preferred embodiment, the gene coding for a transcription factor is a corresponding human-derived gene. As a suitable gene for the transcription factor, the gene for GATA-1 can be named as a transcription factor that binds to the binding sequence (b) GATA. This gene is from Evans et al. in Mol. Cell. Biol., 11 (1991) pp. 843-853. Another suitable transcription factor is called HNF3 and is described by Hafenrichter et al. in Blood 84 (1994), pp. 3394-3404. This transcription factor binds to the binding sequence

GCTCCAGGGAATGTTTGTTCTTAAATACCATGCT.

This gene for the transcription factor can also be part of a chimeric gene, the other part of the chimeric gene coding for a repressor that can bind to the binding site (b). An example of this repressor would be the tetracycline repressor of transposon 10 from E. coli or another repressor that can be regulated by a drug, preferably an antibiotic. The binding site (b) in the nucleic acid construct must then be designed so that the transcription factor or the product of the chimeric gene can bind to the binding site.

According to the invention, the nucleic acid constructs have a host cell-related, constitutively active promoter ("housekeeping promoter"), which is either from the host itself or a closely related species. The host cell-related promoter is preferably the human β-actin promoter.

It is preferred in the context of the present invention that the part of the chimeric gene (d) which codes for a repressor which can bind to the binding site (b) is the tetracycline repressor of the transposon 10 from E. coli. The part of a gene which codes for a transactivation factor, such as, for example, the VP16 protein of the herpes simplex virus, is preferably fused to this.

As a rule, the individual components (a), (b), (c) and (d) are arranged on the polynucleotide in the 5 'to 3' direction.

The nucleic acid constructs according to the invention are generally bare transfectable DNA or plasmid vectors. It is particularly advantageous with the nucleic acid constructs according to the invention that expression can be achieved in the host cells, even if the constructs are not in the form of a viral, in particular retroviral, vector. The use of viral vectors is less preferred in gene therapy due to the unpredictable risks.

The vectors or corresponding nucleic acid constructs can be used without

Origin of replication for the transformation of a eukaryotic host cell, in particular of human cells such as blood stem cells.

By means of the nucleic acid constructs according to the invention, the expression of a foreign gene introduced into the cell can be maintained for a long time, since the host cell does not switch off the expression. The present invention is explained in more detail by the attached figures: FIG. 1A schematically shows monocistronic G-CSF expression constructs. All plasmids are based on pBluescript. ß-Actin is the promoter of the human ß-actin gene. CMV is the immediate-early promoter of CMV (cytomegalovirus). G-CSF is human G-CSF cDNA. TATA is the TATA box. polyA means the small intron and polyadenylation signal of the SV40 virus. tetO means tande -tet operators. The constructs pßACPl.GCSF, pßACP2.GCSF and pßACX.GCSF were generated by inserting tetO trimers at the indicated positions in relation to the TATA box in pßAC.GCSF.

FIG. 1B represents a comparison of the insertion sites of the tet operator sequences within the human β-actin promoter. The tetO trimers were used at the restriction sites indicated in FIG. 1A. The corresponding expression constructs were transiently expressed in KMST-6 cells. G-CSF concentrations in the supernatants were measured 48 hours after transfection by ELISA.

FIG. 2 shows a schematic representation of the dicistronic constructs. All plasmids are based on pBluescript. The abbreviations have the meaning given above. IRES means Internal Ribosome Entry Sequence (of the Encephalomyocarditis Virus) and tetR / VP16 means chimeric transcription activator with the tet repressor and VP16 activity.

Figure 3 shows the kinetics of tetracycline-induced activation and repression of G-CSF production in Balb 3T3 clones that were stably transfected with ptetOtata.GCSF.iresTTAS (one clone) and pßACP2.GCSF. iresTTA

(two clones). The cells were

Cells) and cultured with (tet ⁺ cells) 0.1 μg / ml tetracycline. On day 0, the cells were sown in parallel in cell culture bottles. On each of days 0, 1, 2, 3 and 4 the medium was replaced by an aliquot of the tet ⁺ cells by medium without tet (off-on kinetics) and that Medium from an aliquot of the tef cells was replaced by medium containing 0.1 μg / l tetracycline (on-off kinetics). The appropriate media were renewed on day 4 and G-CSF concentrations in the supernatants were measured on day 5 by ELISA. The differences in the G-CSF scales of the figures may be pointed out.

FIG. 4 shows Table 1, which enables a comparison of the different promoters in the case of transient expression of G-CSF in KMST-6 cells. An expression plasmid for tetR / VP16 (pUHD 15-1) or a control plasmid (pSP65) was co-transfected by cationic lipofection with the indicated G-CSF plasmid in a 1: 1 (weight / weight) ratio. The G-CSF concentrations in the culture supernatants were measured in the absence or presence of 0.1 µg / ml tetracycline 48 hours after transfection with ELISA. The values given correspond to average values of new determinations in ng / ml (± standard deviation).

FIG. 5 shows Table 2, which enables a comparison of the different constructs in the case of transient expression of G-CSF in KMST-6 cells. Equimolar amounts of the plasmids were transfected by cationic lipofection. Appropriate amounts of the control plasmid (pSP65) were added to pßAC.GCSF and pCMV.GCSF to ensure that appropriate amounts of DNA were present in each transfection complex. The G-CSF concentrations in the culture supernatants in the absence or presence of 0.1 μg / ml tetracycline were measured 48 hours after transfection using the ELISA technique. The values correspond to average values of new determinations in ng / ml (± standard deviation).

FIG. 6 shows Table 3, which enables a comparison of the different constructions for the stable expression of G-CSF in Balb3T3 cells. An expression plasmid for Neo ^r (placOSTHNeo) was co-transfected with the indicated G-CSF plasmid in one by cationic lipofection 1: 9 ratio. Stably transfected cells were selected by adding 1 mg / ml G418 to the culture medium. Individual colonies were isolated and expanded. G-CSF concentrations of seven independent clones for each G-CSF expression plasmid were measured in defined culture supernatants in the absence or presence of 0.1 μg / ml tetracycline. Set the values

Mean values in ng / 10 ⁶ cells x 24 hours (± standard deviation). Statistically significant differences were determined by the Wilcoxon's Rank Sum Test between the values marked with an index. The corresponding p-values were ¹ p <0.001, 2p <o θ5, 3p <o, 01.

FIG. 7 shows a comparison of the expression of a vector according to the invention (represented by filled circles) in relation to a corresponding comparison construct (open circles). Recombinant human G-CSF is expressed in mice by the nucleic acid construct according to the invention and the extent of expression is measured by the increase in leukocytes.

The following examples illustrate the present invention:

example 1

Provision of the raw materials

The human ß-actin promoter was taken from a plasmid which contained the ß-actin promoter in a 2.9 kb Sal I - Sac I fragment. The cDNA encoding G-CSF was obtained by RT-PCR amplification of RNA extracted from lipopolysaccharide-stimulated peripheral blood mononuclear cells (plasmids pUHD 15-1 and pUHC 13-3 were prepared according to Gossen and Bujard (PNAS [1992 ], Pp. 5547-5551) The IRES sequence of encephalomyocarditis virus is described, for example, in Zimmermann et al. (Virology [1994], pp. 366-372). The Balb3T3 fibroblast mice were obtained from the ATCC. The immortalized human fibroblast cell lines KMST-6 was developed by Namba et al. (Int. J.Cancer [1985], pp. 275-280). The cell lines were cultured in Dulbecco's modified high glucose Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM sodium pyruvate and 50 μg / ml gentamycin.

The plasmids for the transfections were purified by anion exchange chromatography. Remaining contamination with endotoxin was removed by treatment with polymyxin B. For a standard transfection

10 ^ cells plated in 35 mm plates. The following day, 2 μg of plasmid DNA complexed with 6 μl of the cationic lipofection agent DOSPA / DOPE were added to the cells, under serum-free conditions for 30 minutes. For stable transfection experiments, the plasmids were linearized by cutting with the restriction enzyme Sca I. A mixture of 1.8 μg of the test plasmid and 0.2 μg of placOSTHNeo was combined in the lipofection complex. In order to select stable transfected cells, Gibco's G418 was added to the culture medium 48 hours after transfection, using 0.5 μg / ml for the KMST-6 cells and 1 μg / ml for the Balb3T3 cells.

The determination of the G-CSF concentrations in the culture supernatants was carried out using a commercially available ELIΞA test. In transient expression experiments, the supernatants were collected 48 hours after transfection. In order to generate defined supernatants of the stably transfected clones, 2.5 × 10 ^

Cells sown in 25 cm ^ cell culture bottles. The medium was completely replaced after 48 hours and collected after a further 24 hours. At this point the cells were trypsinized and counted. The Wilcoxon's Rank Sum Test was used for statistical analysis. Human G-CSF was undetectable in supernatants from non-transfected KMST and Balb3T3 cells.

Example 2

The plasmid pCMV.GCSF, shown in FIG. 1A, is based on pBluescript II KS (Stratagene, La Jolla, CA) and contains the human G-CSF cDNA under the control of the immediate / early promoter of CMV and a polyadenylation signal which comes from SV40 . The CMV promoter (control) was replaced by the human β-actin promoter (according to the invention) or the minimal promoter with tetO in order to obtain pßAC.GCSF and ptetOtata.GCSF. The 11th ATG codon of the EMCV IRES from pSport / PV / 2/5 '- Aat was fused to the translation initiation codon of the tetR / VP16 cDNA from pUHD 15-1 using a two-step PCR technology. All fragments generated using PCR technology were checked by DNA sequencing.

Example 3

In a first series of experiments, an attempt was made to identify a site within the human β-actin promoter that would allow the tet operator (tetO) to be inserted without significantly impairing the basic promoter activity. Tandem tetO sequences were obtained by PCR amplification of the tetO heptamer from the plasmid pUHC 13-3 with tetO-specific primers, Pst I or Xho I restriction sites being added at both ends of the PCR products. The polyacrylamide gel electrophoresis of the PCR products showed a ladder of tetO concatamers. tetO trimers were excised from the gel and inserted at the Pst I sites at position -423 and -1663 with respect to the TATA box or at an Xho I site at position -23. These modified promoters were then cloned into pßAC.GCSF instead of the unmodified β-actin promoter. This is shown in Figure 1A. The transient expression of these plasmids in KMST- 6 cells identified the insertion of the tetO trimer at the Pst I site at position -423 (pßACP2.GCSF) as an arrangement that retained 85% of the basic promoter activity. This is shown in Figure 1B. Insertions at other sites or the use of tetO-hexamers led to an at least 65% reduction in promoter activity (FIG. 1B).

Example 4

To test the function of the tetO sequences within the β-actin promoter of pßACP2.GCSF, co-transfection experiments of G-CSF expression constructs were carried out together with an expression construct for tetR / VP16 (pUHD 15-1) in KMST-6 cells. G-CSF expression levels of pßAC.GCSF (no tet operator) corresponded approximately to the level when either pUHD 15-1 or a control plasmid (pSP65) were co-transfected, regardless of the presence or absence of tetracycline (tet) in that Culture medium. This is shown in Table 1 of Figure 4. In contrast, the co-expression of tetR / VP16 in the absence of tetracycline resulted in 3-4 times higher expression of G-CSF from the modified β-actin promoter in pßACP2.GCSF. Addition of 0.1 μg / ml tetracycline to the culture medium or co-transfection of pSP65 instead of pUHD 15-1 reduced the GCSF expression of pßACP2.GCSF to a level comparable to pßAC.GCSF. The tetR / VP16 induced tetO modified β-actin promoter was also stronger than the fully induced minimal tetO promoter PCMV * -1 *. This construct corresponds to ptetOtata.GCSF, shown in FIG. 1A. The results confirm the function of the tetO sequences within the ß-actin promoter and clearly show the ability of the tetR / VP16 system of transcriptional regulation in the cell lines used according to the invention. However, all of these promoters resulted in a significantly lower expression compared to the strong immediate-early CMV promoter in pCMV.GCSF. Corresponding results were obtained in Balb3T3 cells. Example 5

After an optimal promoter arrangement was determined, dicistronic expression plasmids were constructed for the simultaneous expression of a therapeutic gene and tetR / VP16. The translation initiation codon of the tetR / VP16 cDNA was fused to the 11th ATG codon from EMCV in a two-step PCR procedure and the IRES-tetR / VP16 sequence obtained was inserted downstream of the G-CSF cDNA in pßAC.GCSF , pßACP2.GCSF and ptetOtata.GCSF. This is shown in Figure 2.

Example 6

The dicistronic vector was compared to the monocistronic G-CSF expression plasmid in transient expression experiments in KMST-6 cells. To standardize this experiment for the difference in the size of the expression vector, pCMV.GCSF and pßAC.GCSF were mixed with appropriate amounts of pSP65 to maintain equimolar ratios of the expression vector within the 2 µg DNA used for standard transfection complexes. In this normalized experiment, the CMV promoter was about three times stronger than the wild-type β-actin promoter. This is shown in Table 2 of Figure 5. The feedback transcription activator arrangement with the tetO-modified β-actin promoter (pßACP2.GCSF. IresTTA) led to even higher expression levels compared to the strong CMV promoter. In contrast to the cotransfection experiments with monocistronic vectors (Table 1), the transcription of pßACP2.GCSF. iresTTA only partially inhibited by the addition of tetracycline.

In addition to transient expression, the expression levels of the feedback activating vector were also examined after integration into the cellular genome. Balb3T3 cells were co-transfected with one of the dicistronic Plasmids and placOSTHNeo. Individual neoycin-resistant colonies were isolated and expanded and defined supernatants were examined for the production of G-CSF. This is shown in Table 3 of Figure 6. The tetO - ires- tetR / VP16 arrangement resulted in an average three-fold increase in G-CSF production compared to the conventional ß-actin promoter (p <0.001). This increase was partially inhibited by tetracycline if it was present during the subculturing of the cells for the collection of the defined supernatants. The co-expression of tetR / VP16 from a wild-type ß-actin promoter (pßAC.GCSF. IresTTA) led to a transient but also variable expression that was statistically different from ρßACP2.GCSF. iresTTA (ρ <0.01) and pßAC.GCSF (p <0.05) regardless of the presence or absence of tetracycline. The strongest production of G-CSF was in a pßACP2.GCSF. iresTTA-transfected KMST-6 clone measured and amounted to 2.4 μg G-CSF per 10 ⁶ cells in 24 hours.

Example 7

In addition to these constitutively active promoters, an analog expression plasmid based on the minimal tetR / PV16-inducible promoter was examined (ptetOtata. GCSF. IresTTA) (shown in FIG. 2). In stable transfection experiments with both cell lines, an average production of G-CSF was found in the absence of tetracycline, more than two orders of magnitude below the feedback construct with the modified ß-actin promoter. This is shown in Table 3. The transgene expression was therefore highly variable between individual, with ptetOtata .GCSF. iresTTA transfected clones.

To the full regulatory potential of tetracycline for G-CSF expression of pßACP2.GCSF. iresTTA and ptetOtata. GCSF. To investigate iresTTA, the "on-off" and "off-on" kinetics were studied in representative Balb3T3 clones with a high production of G-CSF for the corresponding plasmid. This is shown in Figure 3. In both cases, maximum and minimum expression levels were obtained five days after the removal or addition of tetracycline. While the minimal promoter plasmid allowed the regulation of G-CSF by a factor of five, the modified ß-actin promoter could only be repressed by about 40%.

Example 8

Evidence of longer-term expression

In order to demonstrate that a longer-term expression of a transgene in vivo is made possible by the nucleic acid constructs according to the invention, the two vectors pßACP2tetO.GCSF. irestTA (according to the invention) and pßAC.GCSF (comparison) compared.

Mice were Balb3T3 cells either with the expression vector pßACP2tetO according to the invention. GCSF. irestTA or with the comparison vector pßAC.GCSF (without a binding site for a transcription factor and without a gene coding for a transcription factor), each in combination with an expression construct for neomycin phosphotransferase transfected by cationic lipofection. Stable transfected clones were isolated by selection with G418 and then expanded. One Balb3T3 clone of each plasmid was selected for in vivo experiments.

The one with pßACP2tet .GCSF. As a therapeutic gene product, irestTA transfected Balb3T3 clone produces approximately 1.2 μg of recombinant human G-CSF per 24 h and 10 ^ cells. The Balb3T3 clone transfected with the reference construct (pßAC.GCSF) produces approximately 0.25 μg of recombinant human

G-CSF per 24 h and 10 ⁶ cells.

5 10 ^ cell SCID-type mice were injected subcutaneously from each of the two clones. Per plasmid or clone 3 mice each used. The leukocyte count of the mice was then determined as a measure of the expression of the therapeutic genes.

As shown in Figure 7, the use of the nucleic acid construct according to the invention pßACP2tetO. GCSF. irestTA to a marked and permanent increase in leukocytes, while using the reference vector pßAC .GCSF. irestTA only led to a minimal and temporary increase in leukocytes.

Claims

claims

1) Nucleic acid construct which is suitable for the expression of a foreign gene in a host cell, characterized in that it has the following components:

(b) at least one binding site for a transcription factor,

(c) at least one transgene and

(d) at least one gene coding for a transcription factor which can bind to the binding site for a transcription factor (b).

2) Nucleic acid construct according to claim 1, characterized in that the gene coding for a transcription factor (d) is selected from the GATA-1 transcription factor and the transcription factor HNF3.

3) Nucleic acid construct according to claim 1, characterized in that the binding site for a transcription factor (b) is the binding site for a repressor and that the gene coding for the transcription factor (d) is a chimeric gene coding for is a repressor that can bind to the binding site for a repressor (b), fused with at least part of a transactivation factor. 4) Nucleic acid construct according to claim 3, characterized in that the binding site for a repressor (b) is the tet operator.

5) Nucleic acid construct according to one of claims 1 to 4, characterized in that the binding site for the transcription factor (b) is present several times in a tandem-like arrangement.

6) Nucleic acid construct according to one of the preceding claims, characterized in that an internal ribosome entry sequence (IRES) is arranged between the transgene (c) and the gene coding for the transcription factor (d).

7) Nucleic acid construct according to claim 6, characterized in that the internal ribosome entry sequence is the IRES of the encephalomyocarditis virus.

8) Nucleic acid construct according to one of the preceding claims, characterized in that it is in the host cell-related, constitutively active promoter (a) is the human ß-actin promoter.

9) Nucleic acid construct according to one of claims 3 to 8, characterized in that the part of the chimeric gene (d) which codes for a repressor which can bind to the binding site (b) is the tetracycline repressor of the transposon 10 from E. coli .

10) Nucleic acid construct according to claim 9, characterized in that the part of the chimeric gene which codes for a transactivation factor is the VP16 protein of the herpes simplex virus.

11) Nucleic acid construct according to one of the preceding claims, characterized in that the individual Components (a), (b), (c) and (d) are arranged in the 5 'to 3' direction.

12) Nucleic acid construct according to one of the preceding claims, characterized in that it is a vector.

13) Nucleic acid construct according to claim 12, characterized in that it is a plasmid vector.

14) Use of a nucleic acid construct according to any one of claims 1-13 for the transformation of a eukaryotic host cell.