DE102006011918A1 - New recombinant nucleic acid, useful for controlled expression of foreign sequences, includes a regulatory unit, the tet-operator and an independent origin of replication - Google Patents

Abstract

The invention relates to a recombinant nucleic acid molecule for expressing at least one foreign nucleic acid, the transcription of which is under the control of at least one regulatory unit. The invention further relates to a method for expressing at least one foreign nucleic acid, in which the foreign nucleic acid is cloned into the nucleic acid molecule. The recombinant nucleic acid molecule comprises at least one operator (tet operator) for binding a tetracycline repressor molecule, which is positioned between the sequence segment comprising the foreign nucleic acid and the regulatory unit, and at least one origin of replication (oriP) for independent replication of the nucleic acid molecule.

Description

The The invention relates to a recombinant nucleic acid molecule for expression at least a foreign nucleic acid, whose transcription is under the control of at least one regulatory Unit stands. The invention further relates to a method for expression at least one foreign nucleic acid, in which the foreign nucleic acid cloned into the nucleic acid molecule becomes.

Of the classical approach to heterologous protein expression in mammalian cell lines based on plasmids (e.g., pcDNA3) containing a cDNA downstream of carry a strong viral promoter. As part of a stable Transfection under antibiotic selection only survive the cells that integrate the plasmid into their genome. In the surviving cells that will desired Protein produced permanently (= constitutively). The constitutive Expression is basically unfavorable, for example, if the protein is toxic to the cell, which in extreme cases already during the selection phase leads to death of the desired cells. A controllable expression avoids this disadvantage. Here is the expression first is switched off and only on demand by adding an inductor switched on. There are several systems for controlled expression of proteins in mammalian cells. Widely used is the Tet system, which is based on the tetracycline repressor. In the simplest case (Yao et al., 1998), the Tet repressor binds to its Operator sequence, thus blocking the transcription of a downstream cDNA. By addition of tetracycline or its analogue Doxyzklin in the culture medium, the expression is turned on, wherein the inductor binds to the repressor, who then releases the operator. complicated Variants were developed by Bujard and colleagues (Tet-On and Tet-Off) (Gossen et al., 1994; Gossen and Bujard, 1992). Explicit benefits This controllable expression, first, the possibility of dosing the Expression through change the concentration of the inductor and secondly a homogeneous background. In the case of permanent expression must however, two separate cell lines (with (= vector plus cDNA) and without (= Empty vector) expression of the cDNA) can be applied to the function of the protein. While of the selection doing secondary changes arise, which abolish the comparability of the cell lines.

In the case of the Flp-In ^™ -T-REx ^™ system (short FIT system) from Invitrogen, a plasmid is integrated in the first transfection into the genome which carries an FRT sequence (= FLP recombinase target site). In the second transfection, an expression plasmid for the Tet repressor is integrated at another site in the genome. Finally, in the third transfection, two plasmids are transfected simultaneously, pOG44, an expression plasmid for the FLP recombinase, and pcDNA5 / FRT / TO plus cDNA. The transiently expressed recombinase inserts the pcDNA5 plasmid into the genomic FRT site. The resistance gene on the pcDNA5 plasmid lacks the start codon, so that upon random integration of the plasmid into the genome no resistant cells arise. Resistant cells are formed only if ordered via the FRT site. This results in homogeneous cell material, so that the clone singulation and testing of individual cell clones required in other systems (because of the random plasmid integration at different positions in the genome) is not required here. However, it is a disadvantage of this system that the transfection of a cell line takes 10 weeks (all 3 transfections in succession, including each clonal separation after the first two transfections) or at least 3 weeks are needed if the cell line is purchased after the first two transfections, which in turn significantly increases costs. The decisive disadvantage of the known systems, however, lies above all in the fact that a successful recombination is a rare event, so that the efficiencies of the transfections are very low.

Out WO 99/00510 A1 discloses a method and a controlled vector Expression of a foreign DNA gene in mammalian cells known, the Transcription by the tetracycline inducible tetracycline resistance operator (tet operator) is controlled. The recombinant vector contains a mammalian promoter with a TATA element, at least one tet operator and the foreign DNA gene. This vector either additionally contains the gene for the tetracycline repressor (Tet repressor) or it is introduced into cells containing the tetracycline repressor protein (tet repressor) constitutively express. By adding tetracycline then the expression of the foreign protein can be induced. The Here used viral vectors integrate into the genome the host cells, d. H. it is a clonal separation required and the efficiency of the process is very low.

It Object of the invention, a nucleic acid and a method of the initially mentioned type available to provide a fast and effective protein expression.

The object is achieved by a recombinant nucleic acid molecule, the at least one operator (tet operator) for binding a tetracycline repressor molecule, which is positioned between the sequence comprising the foreign nucleic acid sequence and the regulatory unit, and at least one replication origin (oriP) for independent replication of the nucleic acid molecule.

• a) Klonieren der Fremd-Nukleinsäure in das erfindungsgemäße Nukleinsäuremolekül,
• b) Einbringen des Nukleinsäuremoleküls in geeignete Zellen,
• c) Stabile, episomale Vermehrung des Nukleinsäuremoleküls in den Zellen und
• D) Inkubation der transfizierten Zellen mit einer Substanz (Induktor), vorzugsweise mit Tetrazyklin oder Doxyzyklin als Induktor, welche die Expression der Fremd-Nukleinsäure induziert.

The object is further achieved according to the invention by a method for expressing at least one foreign nucleic acid which is characterized by the following steps:

• a) cloning of the foreign nucleic acid into the nucleic acid molecule according to the invention,
• b) introducing the nucleic acid molecule into suitable cells,
• c) Stable, episomal propagation of the nucleic acid molecule in the cells and
• D) incubation of the transfected cells with a substance (inducer), preferably with tetracycline or doxycycline as an inducer, which induces the expression of the foreign nucleic acid.

The Nucleic acid molecule according to the invention for example, like the Epstein-Barr virus, stably episomally proliferated, i.e. an integration into the genome does not take place (Yates et al., 1985; Young et al., 1988). While at a classic Integration of 10 million transfected cells only approx. 100 clones result, bringing the success rate at most 0.001%, approx. 1 after transfection of the nucleic acid molecule according to the invention % of the transfected cells stably reproducible (Leight and Sugden, 2001). consequently is that going? inventive method at least 1000 times more efficient than conventional methods. The nucleic acid molecule according to the invention contains all elements for a controllable by tetracycline expression, so that in the inventive method only a single transfection is required. That turns into Overall, a huge time savings, because the transfection of a Cell line takes in the process according to the invention only about 1 week instead of the previous 3 weeks (FIT-System). While the Known systems recombinase-dependent are due to the low recombination probability have a very low efficiency, the process of the invention comes without recombinase, d. H. be with a single transfection all needed Elements are introduced into the cells. The success rate is therefore almost at 100%.

In Advantageous embodiment of the invention is provided that the Nucleic acid molecule according to the invention at least contains a sequence section, the nucleic acid sequence encoding the tetracycline repressor molecule includes. This makes the complete transfection time-saving even then be done in just one step, if the host cell the tetracycline repressor is not constitutively expressed. In this In this case, it is advantageous if the nucleic acid molecule according to the invention is a cassette for expression of the tetracycline repressor molecule (tet repressor). This can, for example, a suitable Promoter and preferably a eukaryotic structural gene, for example a β-globulin intron, to imitate eukaryotic expression. In addition, can to the nucleic acid sequence, which encodes the tetracycline repressor, connect a polyA signal.

In particularly advantageous embodiment of the invention is provided that the sequence portion encoding the tetracycline repressor molecule nucleic acid sequence and the sequence portion encoding a foreign nucleic acid nucleic acid sequence includes, with respect to the direction of transcription oriented the same are. With the same orientation of these two components in the molecule, a effective transfection with high transfection rate and strong subsequent expression of the foreign protein become.

Preferably if the nucleic acid molecule according to the invention is an annular DNA vector, preferably an episomal plasmid. When using a self-replicating, episomal plasmid, for example, for human and primate cells contains specific Epstein-Barr virus components, the inventive method optimally to the human biomedicine mainly used Cell lines are adapted. The size of the plasmids according to the invention, for example, the plasmid pEBTet (e.g., 13.3 kb including cDNA), in Comparison to, for example, pcDNA5 plasmids (e.g., 6.9 kb incl. cDNA) is not problematic, d. H. has no negative effects on the efficiency of the process.

Especially, when the nucleic acid molecule of the invention is an annular DNA vector should be both the cassette for expression of the tetracycline repressor molecule and the regulatory unit for controlling the transcription of the foreign nucleic acid, the tet operator and the foreign nucleic acid comprising Sequence section in the molecule be arranged and oriented so that the transcription respectively in the same direction. In this construction of the vector is advantageously a high transfection rate with strong subsequent expression of the foreign protein guaranteed.

If several, preferably two, tet operators are provided may the expression of the foreign protein in the absence of the inducer practically Completely repressed become.

In Further embodiment of the nucleic acid molecule according to the invention is provided, that the regulatory unit to control transcription the foreign nucleic acid a promoter, preferably the CMV promoter.

Preferably contains the promoter sequence the (or the) tet operator sequence (s).

Preferably is the foreign nucleic acid a cDNA sequence.

The Cassette for expression of the tetracycline repressor molecule preferably a promoter, in particular the RSV LTR promoter, and preferably a gene of a eukaryotic peptide or protein, in particular the rabbit β-globulin intron II.

In Advantageous embodiment of the nucleic acid molecule according to the invention is also provided that oriP the replication origin of Epstein-Barr virus is and / or the molecule additionally a cassette for expression of Epstein-Barr Nuclear Antigen 1 (EBNA-1). This construction of the vector with for human and primate cells specific Epstein-Barr virus components allows the self-reliant episomal Replication of the molecule and at the same time an optimal adaptation of the method according to the invention to those in biomedicine mainly used human cell lines.

• a) rekombinanten Nukleotidsequenz, welche eine Kassette zur Expression einer beliebigen Nukleinsäure, vorzugsweise einer cDNA, mit mindestens einem Operator zur Bindung eines Tetrazyklin-Repressor-Moleküls, eine Kassette zur Expression des Tetrazyklin-Repressors, eine Kassette zur Expression des Epstein-Barr-Nuclear-Antigen 1 (EBNA-1) und einen Epstein-Barr-Virus-Replikationsursprung umfasst;
• b) rekombinanten Nukleotidsequenz, welche eine beliebige Kombination der Nukleotidsequenzen gemäß Seq ID No: 2, 3, 4 und 7 oder der Nukleotidsequenzen gemäß Seq ID No: 2 bis 5 und 7 umfasst;
• c) rekombinanten Nukleotidsequenz gem Seq ID No: 1 oder Seq ID No: 9;
• d) Nukleotidsequenz, welche sich von den Nukleotidsequenzen gemäß a), b) oder c) durch den Austausch zumindest eines Codons gegen ein synony mes Codon unterscheidet;
• e) Nukleotidsequenz, deren komplementärer Strang mit den Nukleotidsequenzen gemäß a), b), c) oder d) hybridisiert;
• f) Nukleotidsequenz, welche zu mindestens 80 %, vorzugsweise 90 %, insbesondere 95 %, mit der Nukleotidsequenz gemäß a), b), c), d) oder e) identisch ist;
• g) Nukleotidsequenz, welche dem komplementären Strang der Nukleotidsequenzen gemäß a) bis f) entspricht.

In particular, the invention also encompasses a nucleic acid molecule having a nucleotide sequence which is selected from the group consisting of a nucleotide sequence

• a) recombinant nucleotide sequence which comprises a cassette for expression of any nucleic acid, preferably a cDNA, with at least one operator for binding a tetracycline repressor molecule, a cassette for expression of the tetracycline repressor, a cassette for expressing the Epstein-Barr nuclear Antigen 1 (EBNA-1) and an Epstein-Barr virus origin of replication;
• b) recombinant nucleotide sequence comprising any combination of the nucleotide sequences according to SEQ ID NO: 2, 3, 4 and 7 or the nucleotide sequences according to SEQ ID NO: 2 to 5 and 7;
• c) recombinant nucleotide sequence according to ID No: 1 or SEQ ID No: 9;
• d) nucleotide sequence which differs from the nucleotide sequences according to a), b) or c) by the exchange of at least one codon for a synonymous mes codon;
• e) nucleotide sequence whose complementary strand hybridizes with the nucleotide sequences according to a), b), c) or d);
• f) nucleotide sequence which is identical to at least 80%, preferably 90%, in particular 95%, with the nucleotide sequence according to a), b), c), d) or e);
• g) nucleotide sequence which corresponds to the complementary strand of the nucleotide sequences according to a) to f).

The The invention further encompasses cells containing the nucleic acid molecule of the invention cell cultures and transgenic animals containing these cells and proteins, the means of the nucleic acid molecule according to the invention, the said cells and / or said transgenic animals.

The The invention will be explained below with reference to the figures and exemplary embodiments exemplified in more detail.

In shows the pictures

1 a plasmid map of an embodiment of the nucleic acid molecule according to the invention (pEBTet) without a foreign nucleic acid insert in which the functional elements, their orientation and the cloning sites for the cDNA are marked,

2 a plasmid map of an embodiment of the nucleic acid molecule according to the invention with cDNA insert "SLC22A16" (pEBTet / SLC22A16h) in which the functional elements, their orientation and the cloning sites for the cDNA are marked,

3 A plasmid map for an alternative construct, in which the cassette for expression of the Tet repressor against pEB Tet / SLC22A16h ( 2 ) was turned over (see arrows),

4 a Northern Blot for the analysis of controllability for pEB-Tet / SLC22A16h, wherein the quantitative evaluation was carried out by means of PhosphorImager (Fujifilm BAS-1800 II),

5 a bar graph of the uptake of ergothionein into pEB-Tet / ETTh-transfected cells, wherein the endogenous content of ergothioneine in both cell types was determined by incubation (1 min) with KRH buffer without addition of ergothionein and subtracted from the total uptake in the presence of substrate, and

6 Fluorescence microscopy images for detecting the control of the expression of an ETTh-GFP chimera by pre-incubation with doxycycline, wherein the 293 cells used were transfected with pEB-Tet / ETTh-GFP and selected with puromycin, staining with trypan blue shows the cell membranes of all cells in the field of view.

• 1) pUC-Replikationsursprung und Ampicillin-Resistenzgen für die Vermehrung in E. coli,
• 2) Kassette zur Expression eines Proteins für Puromycin-Resistenz in Mammalia-Zellen (mit SV40-Promotor und Polyadenylierungssignal (SV40 late)),
• 3) Kassette zur Expression des Tetrazyklin-Repressors (Tet-Repressor, Seq ID No: 2; mit RSV-LTR-Promotor, beta-Globin-Intron II (Kaninchen, Seq ID No: 5) und Polyadenylierungssignal (SV40 late)),
• 4) Kassette zur Expression beliebiger cDNAs (hier ohne cDNA-Insert) mit CMV-Promotor (Seq ID No: 8) inkl. 2 × Tet-Operator (Seq ID No: 7), Klonierungsregion (multiple cloning site) und Polyadenylierungssignal (BGH), und
• 5) Epstein-Barr-Virus-Replikationsursprung (oriP, Seq ID No: 3) und Kassette zur Expression von EBNA-1 (Seq ID No: 4).

1 shows an expression vector according to the invention (pEBTet, Seq ID No: 9). This embodiment of a plasmid according to the invention contains the following elements:

• 1) pUC origin of replication and ampicillin resistance gene for propagation in E. coli,
• 2) cassette for expression of a protein for puromycin resistance in mammalian cells (with SV40 promoter and polyadenylation signal (SV40 late)),
• 3) cassette for expression of the tetracycline repressor (Tet repressor, SEQ ID No: 2, with RSV LTR promoter, beta globin intron II (rabbit, Seq ID No: 5) and polyadenylation signal (SV40 late)),
• 4) cassette for expression of any cDNAs (here without cDNA insert) with CMV promoter (Seq ID No: 8) including 2 × Tet operator (Seq ID No: 7), cloning site (multiple cloning site) and polyadenylation signal (BGH ), and
• 5) Epstein-Barr virus origin of replication (oriP, Seq ID No: 3) and cassette for expression of EBNA-1 (Seq ID No: 4).

pEBTet based on the plasmid pCEP-Pu (Kohfeldt et al., 1997). According to the invention was a cassette for the expression of the Tet repressor (Yao et al., 1998) and a cassette for controllably expressing any cDNA added. The Epstein-Barr virus elements oriP and EBNA-1 provide episomal multiplication of the plasmid in human and primate cell lines, i. an integration into the genome does not take place. A clonal singulation is so not required because the plasmid is not integrated into the genome. The selection of the transfected cells is carried out by incubation with puromycin (3 μg / ml). After the selection phase, the expression of the cDNA by addition of tetracycline or doxycycline (1 μg / ml) into the culture medium become. In the ground state (= switched off) there is virtually no mRNA produced.

2 shows the expression vector of the invention according to 1 with cDNA insert (pEBTet / SLC22A16h, Seq ID No: 1). The cDNA of the human protein ETTh (ergothionein transporter, gene symbol SLC22A4, SEQ ID No: 6) was inserted here into the cloning region of pEBTet (Seq ID No: 9).

3 shows an alternative construct in which the cassette for expression of the Tet repressor compared to the vector according to 2 was reversed in terms of its orientation. This construct, when transfected and selected with 3 μg / ml puromycin, yielded very few surviving cells that did not multiply. In contrast, parallel transfection with pEBTet yielded many surviving cells that rapidly multiplied. Consequently, the rectified orientation of the two essential expression cassettes with respect to the direction of transcription for the transfection rate and thus also the expression of the foreign protein is of crucial importance. According to the invention, therefore, the sequence section which comprises the nucleic acid sequence coding for the tetracycline repressor molecule and the sequence section which comprises a nucleic acid sequence coding for the foreign nucleic acid should be oriented identically with respect to the direction of transcription.

4 shows a Northern blot for analysis of controllability for pEB-Tet / SLC22A16h. 293 cells were transfected with the plasmid pEBTet / SLC22A16h. After selection with puromycin, the cells were cultured for about 20 h with or without doxycycline in the medium. The mRNA obtained from both batches was subjected to a Northern blot which was first hybridized with a probe against SLC22A16h and then against GAPDH (control of mRNA loading). The quantitative evaluation was carried out by PhosphorImager (Fujifilm BAS-1800 II). The experiments carried out with the pEBTet vector prove that important favorable properties of the FIT system are retained, ie the controllability (low basal expression, high expression when switched on) corresponds to that of the FIT system. The ratio of SLC22A16h mRNA (switched off) to SLC22A16h mRNA (switched on) is less than 1%, ie the vector according to the invention makes it possible to precisely regulate the expression.

Example 1: Turn on protein expression by pre-incubation with doxycycline increases the uptake of ergothioneine into pEB-Tet / ETTh-transfected cells

The endogenous ergothioneine content of both cell types was determined by incubation (1 min) with KRH buffer without the addition of ergothionein and subtracted from the total uptake in the presence of substrate. Shown in 5 ie the actual uptake during incubation (1 min) with substrate. It is known from other experiments that the nonspecific uptake of ET, eg not mediated by ETT, is negligible due to diffusion.

The cDNA of the human ergothionein transporter ETTh (gene symbol SLC22A4) with a complete open reading frame was inserted into the vector pEBTet (Seq ID No: 9) via the Hin dIII and Not I sites. The resulting construct, pEBTet / ETTh, was transfected into 293 cells, a human embryonic kidney cell line. The cells resulting from the selection with 3 ug / ml puromycin were seeded in poly-ornithine-coated Polystyrolschäl chen (diameter about 6 cm) and incubated for about 3 days. In the last 16 hours prior to the functional assay, half of the dishes were incubated with 1 μg / ml doxycycline to switch on the expression of the transporter. In the functional assay, the sheep were incubated for 20 min in 4.0 ml of KRH buffer (5.6 mM (+) - glucose, 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH ₂ PO ₄ , 1.2 mM CaCl ₂ , 1.2 mM MgSO ₄ and 25 mM HEPES-NaOH pH 7.4) at 37 ° C and then incubated for 1 min with 2 ml of 10 .mu.mol / l ergothioneine (ET) in KRH buffer. The cells were finally washed four times with 4 ml ice-cold KRH buffer to remove extracellular ET, and then digested with 4 mmol / l HClO. ₄ The ET content of the lysates was determined by LC-MS / MS. The evaluation of the data ( 5 ) showed a very low transport activity in the switched-off state (doxycycline minus: 1.8 ± 1.6 pmol min ^-1 mg protein ^-1 ) and a high expression in the on state (doxycycline plus: 46 ± 9 pmol min ^-1 mg protein ^-1 ). Thus, the expression of the transport protein in the cells transfected with the nucleic acid according to the invention could be successfully activated by the inducer doxycycline. The expression of the foreign protein could be carried out very quickly and effectively.

Example 2: Fluorescence detection controlling the expression of an ETTh-GFP chimera by preincubation with doxycycline

The 293 cells used had been transfected with pEBTet / ETTh-GFP and selected with puromycin. Staining with trypan blue shows the cell membranes of all cells in the field of vision ( 6 ). The GFP fluorescence shows in the same image section the expression of the transporter, which is mainly located in the plasma membrane ( 6 ).

The cDNA of the human ergothionein transporter ETTh (gene symbol SLC22A4) was linked at the C-terminus to a cDNA for the green fluorescent protein (E-GFP) in order to be able to produce a chimeric protein. The chimeric cDNA was inserted via the Hin dIII and Not I sites in the vector pEBTet (Seq ID No: 9). The resulting construct, pEBTet / ETTh-GFP, was transfected into 293 cells, a human embryonic kidney cell line. The cells resulting from the selection with 3 μg / ml puromycin were seeded in polystyrene dishes on poly-ornithine-coated coverslips (diameter 12 mm) and incubated for about 2 days. In the last 16 h prior to analysis, half of the coverslips were incubated with 1 μg / ml doxycycline to turn on expression of the GFP-tagged transporter. After washing with 1 × PBS, the cells were stained for 3 minutes at room temperature with 0.02% trypan blue in 1 × PBS. After rinsing again with 1 × PBS, the coverslips containing the unfixed cells were placed on slides and analyzed under the fluorescence microscope ( 6 ). The figure shows virtually no GFP fluorescence in the off state (doxycycline minus) and high expression in the on state (doxycycline plus), ie the expression of the foreign nucleic acid could be carried out by the method according to the invention very quickly and with high efficiency.

Literature:

• Gossen M, Bonin AL, Freundlieb S, Bujard H (1994): Inducible gene expression systems for higher eukaryotic cells. Curr. Opin. Biotechnol. 5: 516-520
• Gossen M, Bujard H (1992): Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89: 5547-5551
• Kohfeldt E, Maurer P, Vannahme C, Timpl R (1997): Properties of the extracellular calcium binding module of the proteoglycan Testican. FEBS Lett. 414: 557-61
• Leight ER, Sugden B (2001): Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event. Mol. Cell. Biol. 21: 4149-61
• Yao F, Svensjö T, Winkler T, Lu M, Eriksson C, Eriksson E (1998): Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum. Gene Ther. 9: 1939-50
• Yates JL, Warren N, Sugden B (1985): Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313: 812-5
• Young JM, Cheadle C, Foulke JS, Jr., Drohan WN, Sarver N (1988): Utilization of Epstein-Barr virus replicon as a eukaryotic expression vector. Gene 62: 171-85

SEQUENCE LISTING

Claims (18)

Recombinant nucleic acid molecule for expression at least a foreign nucleic acid, whose transcription is under the control of at least one regulatory Unit is bound to at least one operator (tet operator) a tetracycline repressor molecule sandwiched between the foreign nucleic acid Sequence section and the regulatory unit is positioned, and at least one replication origin (oriP) for self-replication of the nucleic acid molecule. Nucleic acid molecule according to claim 1, characterized in that it has at least one sequence section contains one encoding the tetracycline repressor molecule nucleic acid sequence includes. Nucleic acid molecule according to claim 2, characterized in that it comprises a cassette for the expression of the Tetracycline repressor molecule (Tet repressor) includes. Nucleic acid molecule according to claim 2 or 3, characterized in that the sequence section, the the nucleic acid sequence encoding the tetracycline repressor molecule and the sequence portion encoding a foreign nucleic acid nucleic acid sequence includes, with respect to the direction of transcription oriented the same are. Nucleic acid molecule according to a the claims 1 to 4, characterized in that this is a circular DNA vector, preferably an episomal plasmid. Nucleic acid molecule according to claim 5, characterized in that the cassette for the expression of the tetracycline repressor molecule and the regulatory unit for controlling the transcription of the foreign nucleic acid, the tet operator and the sequence comprising the foreign nucleic acid sequence in the molecule are arranged and oriented so that the transcription respectively in the same direction. Nucleic acid molecule according to a the claims 1 to 6, characterized in that several, preferably two, tet operators are provided. Nucleic acid molecule according to a the claims 1 to 7, characterized in that the regulatory unit for controlling the transcription of the foreign nucleic acid Promoter, preferably the CMV promoter. Nucleic acid molecule according to a the claims 1 to 8, characterized in that the foreign nucleic acid a cDNA sequence is. Nucleic acid molecule according to a the claims 1 to 9, characterized in that the cassette for expression of the tetracycline repressor molecule Promoter, in particular the RSV LTR promoter, and preferably a gene of a eukaryotic peptide or protein, especially the rabbit β-globulin intron II. Nucleic acid molecule according to a the claims 1 to 10, characterized in that oriP the replication origin of Epstein-Barr virus is and / or the molecule additionally a cassette for expression of Epstein-Barr Nuclear Antigen 1 (EBNA-1). Nucleic acid molecule according to one of claims 1 to 11 comprising a nucleotide sequence which is selected from the group consisting of a) recombinant nucleotide sequence which comprises a cassette for expression of any nucleic acid, preferably a cDNA, with at least one operator for binding a tetracycline repressor molecule, a cassette for expression of the tetracycline repressor, a cassette for expression of Epstein-Barr Nuclear Antigen 1 (EBNA-1) and an Epstein-Barr virus origin of replication; b) recombinant nucleotide sequence comprising any combination of the nucleotide sequences according to SEQ ID NO: 2, 3, 4 and 7 or the nucleotide sequences according to SEQ ID NO: 2 to 5 and 7; c) recombinant nucleotide sequence according to ID No: 1 or SEQ ID No: 9; d) nucleotide sequence which differs from the nucleotide sequences according to a), b) or c) by the exchange of at least one codon for a synonymous codon; e) nucleotide sequence whose complementary strand hybridizes with the nucleotide sequences according to a), b), c) or d); f) nucleotide sequence which is identical to at least 80%, preferably 90%, in particular 95%, with the nucleotide sequence according to a), b), c), d) or e); g) nucleotide sequence which corresponds to the complementary strand of the nucleotide sequences according to a) to f). Cells which contain the nucleic acid molecule according to one of claims 1 to 12 included. Cell culture containing cells according to claim 13. Transgenic animal, which cells according to claim 13 contains. Protein produced by means of the nucleic acid molecule one of the claims 1 to 12, the cells according to claim 13 and / or the transgenic animal according to claim 15. Process for the expression of at least one foreign nucleic acid the following steps: a) cloning the foreign nucleic acid in the Nucleic acid molecule according to a the claims 1 to 12, b) introducing the nucleic acid molecule into suitable cells, c) Stable, episomal propagation of the nucleic acid molecule in the cells and d) Incubation of the transfected cells with a substance (inducer), which induces the expression of the foreign nucleic acid. Method according to claim 17, characterized in that that added to the cells as an inducer tetracycline or doxycycline becomes.