MXPA98009910A - Cell optimization for ge endogenic activation - Google Patents

Abstract

The present invention relates to a process for optimizing gene optimization of cells. A first aspect relates to a process for changing the expression of a nucleic acid sequence which is present endogenously in a eukaryotic cell by induction of a heterologous expression control sequence within the cell genome by means of homologous recombination, as well as a cut site-specific, recombinase-mediated, of the inserted foreign DNA and its replacement of additional heterologous expression control sequences or / and amplification genes. Furthermore, the invention relates to the introduction of one or more nucleic acid sequences to which an activating protein or activating protein complex is linked, for example, a hypoxia inducible factor (HIF) in the genome of a eukaryotic cell by recombination. homologous in order to change the expression of a target gene. In addition, the invention relates to a process for testing the influence of 5'-or 3'-non-coding fragments of nucleic acid on the expression of a target gene by determining the expression of a reporter gene. In addition, the invention relates to a process for providing a DHFR negative eukaryotic cell containing an objective recombinase sequence as well as the expression of a nucleic acid sequence inserted into the target recombinase sequence.

Description

OPTIMIZATION OF CELLS FOR ENDOGENIC ACTIVATION OF GENES PISCRIgCtIon PB AI mvgNCtN The invention relates to a process for optimizing the expression of genes in cells. A first aspect relates to improving the expression of a target gene that is endogenously present in a eukaryotic cell by introducing a heterologous expression control sequence or / and an amplification gene into the cell genome by means of homologous recombination, and it is also related to the cutting of the inserted foreign DNA mediated by a site-specific recombinase and its replacement by other sequences and / or amplification genes of heterologous expression control. The invention is further related to the introduction of one or more nucleic acid sequences to which an activating protein or activating protein complex, for example, the hypoxia-inducible factor (HIF), binds in the genome of a eukaryotic cell. by homologous recombination in order to change the expression of a target gene. In addition, the invention relates to a method for testing the influence of non-coding nucleic acid fragments at the 5 'end or the 3' end on the expression of a target gene by determining the expression of an indicator gene. In addition, the invention relates to a process for preparing a cell REF: 28912 eukaryotic DHFR negative combining an objective sequence for recombinase as well as the expression of a nucleic acid sequence inserted within the target sequence for recombinase. The expression of genes in a cell takes place constitutively, for example, in the so-called maintenance genes, or it can be regulated. Regulated expression is particularly necessary for genes which should only be expressed at a particular stage of cell development or when there is a change in environmental conditions. The expression is regulated at the level of transcription by the promoter that is operatively linked to the coding nucleic acid sequence, activity of which can be controlled by repressors and activators. The binding of repressors or activators to non-coding nucleic acid sequences of the gene can reduce or increase the activity of the promoter (L. Stryer, Biochemie, Chapter 12, "Spektrum der issenschaft, Verlagsgesellschaft", Heidelberg, 1990). The amount of repressors or activators that are contained in a cell in turn is regulated by factors such as, for example, environmental conditions. Hypoxia inducible factors (HIF) are an example of activators which are induced by a reduced supply of 02 and lead to an increased expression of the erythropoietin gene (Blanchard KL et al., Hypoxic induction of the human erythropoietin gene: Cooperation between the promoter and enhancer, each of wich contains steroid receptor response elements, (1992), Mol. Cell, Biol. 12, 5373-5385, Wang GL and Semenza GL, Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia, (1993), J. Biol. Chem., 268, 21513-21518; Wang GL et al., Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PA heterodimer regulated by cellular 02 voltage, (1995) ), Proc. Nati, Acad. Sci. USA, 92, 5510-5514). In addition, the amount of an expressed protein depends on the stability of the mRNA. The recognition sequences for mRNA degrading enzymes are located in the 3 'region of an mRNA which influences the stability of the mRNA and therefore the level of expression (Shaw G. and Kamen, R., A Conserved AU Sequence from the 3'Untranslated Region of GM-CSF mRNA Mediates Selective mRNA Degradation, Cell (1986), 659-667). In this regard, the average length of mRNA is correlated with the amount of protein expressed. A third level of regulation of expression is translation. Therefore, the expression of a gene undergoes complex regulatory mechanisms that can differ widely in individual cases. Proteins can be obtained with the help of recombinant DNA technology which uses knowledge about the regulation of expression (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor).

The vectors that are used for this, which contain a nucleic acid sequence coding for the corresponding protein under the control of a suitable promoter as well as additional sequences that are necessary to express the protein and replicate the vector. In this way the vector is introduced into the host cell by means of known methods, the cell is cultured and the recombinant protein can be isolated from the cell or from the culture medium. Prokaryotic or eukaryotic cells can be used as the host cell. Prokaryotic cells, in particular, E. coli cells are not problematic to manage but have numerous disadvantages when eukaryotic proteins are expressed in a recombinant manner. The prokaryotes and eukaryotes differ in the expression processing trajectories, in the conditions of cellular medium as well as in the chaperones involved in the processing of proteins. Therefore, a eukaryotic protein produced in a prokaryote can differ decisively from the corresponding native protein. For example, the folding pattern of the protein and the activity of the protein can be modified. In addition, proteins in a prokaryotic host cell are usually not glycosylated. However, a correct glycosylation pattern is a crucial feature in many cases for effectiveness and tolerance, for example, in the introduction of proteins for a pharmaceutical formulation. Therefore, glycosylated proteins are produced by means of eukaryotic host cells or cell lines, for example CHO cells (Chinese hamster ovary). Despite the use of these eukaryotic cells, changes in the recombinantly produced protein may occur due to species differences, for example, when a human protein is expressed in non-human cells which is why this method is not suitable for many applications For the recombinant production of proteins, the host cells are transiently or stably transfected with expression vectors, stably transfected cells are used in particular for large-scale production processes. The random and non-specific integration of the expression vector sequences into the host cell genome can lead to cells with a low production capacity or unstable properties of the cells. For example, the yield of production may decrease during the course of a production process, or the ability of the cells to express the recombinant protein may be completely lost. A method for increasing gene expression is the amplification of genes in which the nucleic acid sequence encoding a protein is coupled to an amplification gene. A multiplication of both sequences is obtained by a selection step which leads to an increased expression (Schimke, R.T. (Ed.) (1982), Gene amplification, Cold Spring Harbor Lab., Cold Spring Harbor, NY). For example, a nucleic acid encoding a dihydrofolate reductase (DHFR) can be used as an amplification gene (Kaufmann RJ, Sharp PA (1982), Amplification and expression of sequences contransfected with a modular dihydrofoat reductase complementary DNA gene, J. Mol. Biol. 159: 601 ff). A selection step carried out with methotrexate allows obtaining cells which are resistant to methotrexate and which contain in their genome the nucleic acid sequence encoding a DHFR and the nucleic acid sequence coupled thereto, with a amplification 20 to 50 times (R. Knippers, 1982, "Molekulare Genetik", Thieme, Stuttgart). Such a method of gene amplification is carried out more effectively with a negative DHFR cell. JP-62265992 describes, for example, a human DHFR cell negative. However, a site-specific integration of an expression vector by means of homologous recombination and amplification of these sequences in this cell is not mentioned. Even when a gene amplification process is carried out, the disadvantages described above such as instability of the cells due to random integration of the expression vector into the genome of the cell can be presented. It is only possible to avoid the disadvantages described when the foreign DNA is specifically integrated into the site at a selected gene locus by homologous recombination which leads to an endogenous activation of the gene. Corresponding methods are known and are referred to as gene insertion (WO 90/11354; WO 91/09955). In this process the cell is transfected with a vector which contains a positive selection marker gene flanked by nucleic acid sequences which are homologous to a gene locus in which an attempt is made to integrate the vector into the genome of the cell. Among the homologous nucleic acid sequences there is additionally a heterologous expression control sequence in order to increase the expression of the target gene in the cell and optionally an amplification of the gene to increase the copy number of the target gene. A disadvantage of previously known gene insertion methods is that they are often extremely laborious to produce cells with properties that allow the production of a suitable protein in an amount and quality suitable for commercial purposes. In particular, the selection of the optimal expression control sequences and / or the amplification genes for the expression of a desired target protein often requires a very large series of homologous recombination experiments which take up too much time due to the complexity of the procedure for isolating clones in which the desired recombination element has taken place. Homologous recombination can also be used to inactivate the expression of certain genes in a cell in order to carry out protein function studies. For this purpose, agénic mice are produced in which the gene coding for a protein to be examined has been eliminated by homologous recombination in embryonic pluripotent cells. After carrying out the additional processing steps, mice are obtained that can not express a functional protein from the beginning of its development due to the inactivation of both alleles of this gene (Thomas, KR, Capecchi MR, (1987), Site -directed mutagenesis by gene targeting in mouse embryo-derived stem cells, Cell 51: 503-512). The Cre-Lox system can be used to inactivate a specific gene specifically and in time in tissue and to examine it. For this purpose, a nucleic acid fragment flanked by two loxP sequences is introduced into the genome of a cell by homologous recombination and subsequently can be separated again from the genome by a Cre recombinase that is expressed in the cell (Sauer B, Henderson N ( 1989): Site-specific DNA recombination at loxP sites placed in the genome of mammalian cells Nuc Acid Res 17: 147-161; Sauer B., Henderson N. (1990), Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase, New Biol. 5: 441-449). The prior art makes no mention of using the Cre-lox system in another site-specific recombinase system for the site-specific integration of expression control sequences or amplification genes within the genome of eukaryotic cells in order to change the endogenous expression of the gene. The aim of the present invention is to provide a novel process for optimizing endogenous gene activation by homologous recombination which at least partially eliminates the disadvantages of the prior art. This objective is obtained by providing a new vector process and constructs which considerably simplify the optimization of gene expression performance in eukaryotic cells. A first aspect of the invention relates to a process for changing the expression of a nucleic acid sequence which is presented endogenously in a eukaryotic cell which is characterized in that (a) the cell is transfected with a first vector comprising: i) at least one sequence that is selected from a first heterologous expression control sequence and a first amplification gene, (ii) a positive selection marker gene, (iii) at least two target sequences for site-specific recombinase. that flank the sequences (i) (ü), (iv) DNA sequences which flank sequences (i), (ii) and (iii) and which are homologous to the nucleic acid section in the genome of the cell in order to allow homologous recombination (b) ) the transfected cell is cultured under conditions which homologous recombination of the vector takes place, and (c) the cell obtained according to step (b) is isolated. A cell is provided by the process according to the invention which has an endogenous gene in operable linkage with a heterologous expression control sequence or / and an amplification gene, and these sequences are flanked by target sequences for site-specific recombinase. , for example, Cre recombinase. This cell is very suitable for investigations regarding the optimization of expression of the target gene since the presence of the target sequences for the site-specific recombinase allows for a simple substitution of the first heterologous expression control sequence or / and the first heterologous expression gene. amplification by a second heterologous expression control sequence or / and a second amplification gene. The term "site-specific recombinase" according to the present invention encompasses proteins and protein complexes which mediate rearrangements of DNA in a specific DNA target sequence that includes site-specific recombinases of the integrase or resolvase invertase classes (Stark et al. al., Trends Genet 8 (1992), 432-439; Abremski and Hoess, Protein Engineering 5 (1992), 87-91; Khan et al., Nucleic Acids Res. 19 (1991), 851-860) and site-specific recombination mediated by intron encoded endonucleases (Perrin et al., EMBO J. 12 (1993), 2939-2947). Preferred recombinase proteins are selected from the group comprising FLP recombinase of the 2 μm episome of Saccharomyces cerevisiae (eg Falco et al., Cell 29 (1982), 573-584; Cox, Proc. Nati. Acad. Sci. EUA 80 ( 1983) 4223-4227; Konsolaki et al., New Biologist 4 (1992), 551-557), the Cre recombinase of phage Pl with E coli (for example Sauer and Henderson (1989) supra), the R recombinase for the plasmid pSRl from Zygosaccharomyces rouxii (Matsuzaki et al., J. Bacteriol 172 (1990), 610-618), recombinase A for plasmid pKDl from Kluyveromyces drosophilary (Chen et al., Nucleic Acids Res. 14 (1986), 4471 -4481), recombinase A from the plasmid pKW1 of Kluveromyces waltii (Chen et al., J. Gen. Microbiol. 138 (1992), 337-345), a recombination system component? -int (Landy, Annu Rev. Biochem.5 (1989), 913-949) and a component of the gin recombination system of phage μ (Klippel et al., EMBO J. 12 (1993), 1047-1057). In addition, the fusion proteins described in European patent EP-B-0 707 599 consisting of a site-specific recombinase and a nuclear receptor or the ligand-binding domain thereof are also suitable. The target sequences of the Cre recombinase, i.e., the loxP sequences, are preferably used for the process according to the invention. In contrast to the recombinant production of proteins by non-specific site integration of heterologous genes and their associated disadvantages, the process according to the invention utilizes the advantages of site-specific endogenous gene activation by homologous recombination. A simplified selection of suitable combinations of heterologous expression control sequences and amplification genes allows optimized production clones with stable properties to be obtained, with a high probability, which allows the production of a protein which substantially corresponds to the native protein with respect to its structure and activity. The selection of suitable homologous sequences, which flank the heterologous expression control sequence, the amplification gene, the positive selection marker gene and the recombinase target sequences is preferably carried out according to the method described in WO90 / 11354 and WO91 / 09955. In addition, the homologous sequences may also contain modifications which lead to mutations in the expressed protein such as, for example, point mutations, insertions or / and deletions of individual amino acids or of whole sections of amino acids. Therefore, the process according to the invention not only allows the level of expression of a nucleic acid sequence to be changed in a single process step, but also simultaneously allows the introduction of a mutation in the coding region of the invention. the endogenous nucleic acid sequence. Therefore, the process according to the invention is particularly advantageous for the production of proteins for pharmaceutical applications. Such proteins may have no additional modifications compared to the native proteins in addition to the mutations to increase the efficiency of the protein. According to the invention, it is possible to use any eukaryotic cell, preferably a mammalian cell, particularly preferably a human cell. The process according to the invention can be carried out with non-immortalized cells, for example, fibroblasts and also with immortalized cells, for example, tumor cell lines. Immortalized cells are preferred. The solutions and means used to carry out the process according to the invention are preferably selected so that optimum conditions are present in the respective process step. The cells are cultured using medium which contains all the substances necessary for adequate cell growth and optionally is buffered. It is preferable to use cells which can be cultured in a serum-free medium. The cell used is particularly preferable a Namalwa cell, HT1080 or HeLa S3. The process according to the invention allows the optimization of the expression of a nucleic acid sequence present endogenously in the cell, ie of a target gene by selection of an optimal expression control sequence, an optimal amplification gene or / and by selection of an optimal combination of expression control sequence and amplification gene. Any nucleic acid sequence can be used as the heterologous expression control sequence which influences the expression of the target gene after its integration into the genome of the cell. This includes nucleic acid sequences which can interact directly with transcription components such as transcription start factors or RNA polymerases and nucleic acid sequences whose influence on transcription is mediated by interactions with activators or repressors. The heterologous expression control sequence preferably contains a promoter / extender, particularly preferably viral promoters and most preferably a CMV promoter. The heterologous expression control sequence may also include a 3 'non-coding sequence. The 3 'non-coding sequences may have a stabilizing or destabilizing effect on an mRNA and therefore increase or decrease its average duration. The introduction of a sequence that stabilizes an mRNA can increase the average length of an mRNA and therefore the performance of its encoded protein. - - - - -. In a preferred embodiment, a control sequence for endogenous expression of the target gene is removed by homologous recombination. This is particularly advantageous when the endogenous sequence contains a repressor binding sequence. Expression can also be reduced by a 3 'non-coding sequence which has a destabilizing effect on the mRNA which results in a decrease in the amount of translated protein. In addition, the process according to the invention allows the selection of an optimal amplification gene. The amplification gene is preferably used in an expressible form, that is, in operative binding with a suitable promoter and placed in the vector so that after the homologous integration of the vector into the genome of the eukaryotic cell, it is located near the gene objective. Performing an amplification step leads to an increase in the number of copies of the target gene in the cell. This may result in a further increase in the expression of the endogenous nucleic acid sequence. Examples of suitable amplification genes are dihydrofolate reductase (DHFR), adenosine deaminase, orinitine decarboxylase or muteins of these genes. The amplification gene preferably is a gene for DHFR or an adopted form thereof (Simonsen et al., Nucleic Acids Res. 1988, 16 (5): 2235-2246), especially in cells which contain an endogenous gene for DHFR. Any suitable resistance gene for a eukaryotic cell, which leads to a selectable phenotype such as, for example, antibiotic resistance, can be used as a positive selection marker. The positive selection marker gene is preferably a gene that provides resistance to neomycin, kanamycin, genemycin or hygromycin. Preferably, the positive selection marker gene is used in an expressible form, that is, in operative binding with a suitable promoter. If a negative selection marker gene is used, then a second negative selection step is usually carried out in addition to the positive selection step. The advantage of this is that, after carrying out the selection steps, the identified clones contain a smaller proportion of false positive clones, ie, vectors that are randomly integrated into the genome. The negative selection marker gene is preferably a gene for thymidine kinase (TK) or / and a gene for hypoxanthine-guanine-phosphoribosyl transferase (HGPRT). As a result of the presence of target sequences of site-specific recombinase, it is possible to cut nucleic acid sequences located between these sequences of the cell genome using the site-specific recombinase. The nucleic acid sequence located between the target sequences is preferably cut from the genome by transient activation of the corresponding recombinase in the cell. This transient activation of the recombinase can, for example, be carried out by: (a) transfecting the cell with a second vector containing a nucleic acid sequence encoding the recombinase, operably linked to an expression control sequence that is active or that can be activated in this cell, and (b) culturing the transfected cell in this manner under conditions under which the recombinase is expressed and active, and (c) optionally isolating the cell. If the recombinase / nuclear receptor fusion proteins are the ones used, the transient activation of the cell can also be carried out by the controlled addition of the ligand to the nuclear receptor. After removing the localized DNA between the target sequences, the remaining target sequence, for example, the loxP sequence can be used for additional process steps. In a further preferred embodiment, the process is characterized in that: (a) the cell is transfected with a third vector comprising (i) at least one sequence that is selected from a second heterologous expression control sequence and a second gene from amplification (ii) a positive selection marker gene which preferably differs from the positive selection marker gene of the first vector, and (iii) at least two target sequences for recombinase bleaching sequences (i) and (ii) (b) ) the transfected cell is cultured under conditions under which the sequence flanked by the target sequences is integrated into the target sequence in the genome of the cell (c) the cell obtained is isolated according to step (b), and ( d) steps (a) to (c) are optionally repeated at least once with expression control sequences or / and amplification genes which vary, in each case. Therefore, the process according to the invention allows many expression control sequences, amplification genes or combinations of expression control sequences and amplification genes to be tested simply and quickly. Therefore, it is not necessary to perform site-specific, time-consuming and costly integration for each individual heterologous expression control sequence for each individual amplification gene in order to determine an optimal expression / amplification system for each individual target gene. The positive selection marker gene in a third vector preferably differs from a first vector in order to simplify the selection process and to minimize the number of false positive clones. The target recombinase sequences in the vector used according to the invention may correspond to target sequences that occur naturally or optionally have mutations which do not alter the effectiveness of the site-specific recombinase. A further subject matter of the invention is a vector for homologous recombination in particular for site specific introduction of recombinase target sequences within the genome of a cell, comprising: (i) at least one sequence that is selected from a sequence of expression control and an amplification gene, (ii) a positive selection marker gene, (iii) at least two target sequences for a site-specific recombinase which flank sequences (i) and (ii) (iv) DNA sequences flanking sequences (i), (ii) and (iii) which are homologous to a section of nucleic acid in the genome of a cell in order to allow homologous recombination, and (v) optionally a negative selection marker gene. In addition, all vectors according to the invention preferably contain the sequence elements necessary for propagation and multiplication in suitable host cells such as origin of replication, selection marker genes, etc. A further subject matter of the present invention is a vector, in particular for introducing DNA into the genome of a cell by means of a site-specific recombinase system, comprising: (i) at least one sequence that is selected from a sequence of expression control and an amplification gene, (ii) a marker gene of positive selection, and (iii) at least two target sequences for recombinase flanking sequences (i) and (ii). A further subject matter of the present invention is a eukaryotic cell, preferably a human cell, which can be obtained by a process as described above. This cell, e.g., a human cell, is preferably characterized in that: (a) it contains at least one chromosomally located sequence that is selected from a heterologous expression control sequence and an amplification gene in operable linkage with an acid sequence nucleic that is endogenously present, and (b) this sequence is flanked by target sequences for recombinase. A further aspect of the present invention relates to a process for changing the expression of a nucleic acid sequence that is endogenously present in a eukaryotic cell, which is characterized in that: (a) the cell is transfected with a vector comprising: (i) at least one nucleic acid sequence that binds to an activating protein, eg, a hypoxia-inducible factor, (HIF), (ii) a positive selection marker gene, (iii) flanking DNA sequences to sequences (i) and (ii) which are homologous to a section of nucleic acid in the genome of the cell in order to allow homologous recombination, (b) the transfected cell is cultured under conditions under which it takes place the homologous recombination of the vector, and (c) the cell obtained according to step (b) is isolated. Surprisingly, the genomic integration of a nucleic acid sequence which binds to one or more activating proteins (proteins which increase the expression of the gene by binding to the nucleic acid sequence) in the region of the expression control sequence of a particular target gene in its regulatory regions, does not reduce the expression of the target gene but, in contrast, it is possible by selecting suitable culture conditions to increase the expression of the endogenous target gene or to induce the expression of an endogenous target gene not voiced. Examples of suitable activating proteins are hypoxia-inducible factors HIF-lo- and HIF-lβ, as well as interferon-regulated factor 1 (IRF-1) which can increase transcription by binding to the interferon consensus sequence ( ICE) (Tanaka N., Kawakami T., Taniguchi T., Mol. Cell. Biol. (1993), August; 13 (8): 4531-4538). After operative binding of one or more nucleic acid sequences that bind to HIF or other activator proteins to a target gene that is endogenously present, the expression of the target gene can be regulated by selecting suitable culture conditions. An advantage of this, especially for commercial scale production, is that the expression of a protein can be induced at an optimum time for production processes. This is beneficial because the average residence time of the synthesis product in the supernatant of the culture medium is reduced. The amount of undesired degradation products of the protein is also reduced. This is a positive effect in the subsequent purification stages, reduces production costs and leads to a qualitatively improved final product. In order to carry out the process according to the invention, it is sufficient to operatively link one or more nucleic acid sequences that bind to the activator with the target gene. Preferably, two nucleic acid sequences that bind to HIF are used. The nucleic acid sequence that binds to HIF is preferably selected particularly from the 53 bp sequence according to SEQ. FROM IDENT. NO.l, the 43 bp sequence according to SEC. FROM IDENT. NO.2, a sequence homologous to these sequences or a sequence that hybridizes with these sequences under restriction conditions. The use of two nucleic acid sequences that bind to HIF surprisingly leads to a synergistic effect. This leads to a greater increase in the expression of endogenous nucleic acids than when each of these sequences is used alone. If necessary, the expression of the activating protein which binds to the activating sequences introduced in the region of the target gene can be induced or increased in the cell. This can be obtained, for example, by transfecting the cell with a vector, comprising: (i) a nucleic acid sequence encoding an activator protein which is operably linked to an active expression control sequence in this schedule, and (ii) optionally a positive selection marker gene. Any nucleic acid sequence encoding an activating protein can be used whose expression product can be linked to a nucleic acid sequence that binds to an activator integrated into the genome. The activator protein preferably is a HIF-la or / and HIF-ljß protein. If the nucleic acid sequence that is present endogenously already contains nucleic acid sequences that bind to activator or preferably that bind to HIF it may be sufficient to only introduce a vector into the cell containing a nucleic acid sequence encoding a activator protein or preferably for a HIF protein which is operably linked to an active expression control sequence in the cell and optionally with a positive selection marker gene. The expression control sequence which is operably linked to the nucleic acid sequence encoding the activating protein may be inducible, which provides an additional method for activation by suitable culture conditions such as, for example, by the addition of hormones. or heavy metals. This allows the expression of an endogenous target gene that is to be induced at an optimum time for the production process. An advantage of using a constitutively active expression control sequence is that the activating protein is constitutively expressed independently of the addition of activators in the culture medium. If the nucleic acid sequence that binds activator protein is a nucleic acid sequence that binds to HIF, the expression of the target gene can, for example, be induced by suitable culture conditions., for example, at a concentration of 02 of 0.1-2%. A further subject matter of the present invention is a vector for homologous recombination, comprising: (i) at least one nucleic acid sequence which binds to an activating protein, (ii) a positive selection marker gene, (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a section of nucleic acid in the genome of a cell in order to allow homologous recombination.

A further subject matter of the present invention is a eukaryotic cell, preferably a human cell, which can be obtained by one of the processes described above. This cell preferably is characterized in that it contains at least one fragment of nucleic acid that binds to the activating protein complex, located chromosomally, heterologous, operationally linked to a gene that is present endogenously in the cell. The nucleic acid fragments that bind to activating protein can be substituted in the genome with the help of a site-specific recombination system as elucidated in the above, which allows a simple identification of an optimal activating sequence for a certain target gene. . A further aspect of the present invention relates to a process for testing the influence of its expression of non-coding nucleic acid sequences from the region of a target gene endogenously present in a eukaryotic cell, which is characterized in that: (a) The cell is transfected with a vector comprising: (i) a heterologous expression control sequence that is active or that can be activated in the cell, which is operably linked to a reporter gene, and (ii) non-coding acid fragments. nucleic at the 5 'or / and at the 3' end of the target gene region, (b) the cell is cultured under conditions under which the expression control sequence is activated, and (c) expression is measured of the indicator gene. The way in which a heterologous expression control sequence should be placed in the region of the target gene in the genome can be determined in a simple manner with the process according to the invention, in order to obtain an optimal expression rate of the gene objective and what influence has the presence or absence of 5 'or / and 3' non-coding sequences of the region of the target gene on expression. The test vectors are preferably transiently transfected into cells and expression of the reporter gene is determined. In this way, it is possible to quickly and inexpensively test many arrays or arrangements of a heterologous expression control sequence and a target gene, or many different expression control sequences. The heterologous expression control sequences include nucleic acid sequences which can directly interact with transcription components such as transcription initiation factors or RNA polymerases and nucleic acid sequences whose influence on transmission is mediated by interactions by activators or repressors. The heterologous expression control sequence is preferably a promoter / extender, particularly preferably a viral promoter and more preferably a CMV promoter. The process according to the invention contributes to a strong reduction of costs especially in processes which contain additional steps with complicated process. This is the case, for example, in the production of transgenic animals such as mice, sheep or cows, in which it is intended to increase the expression of a particular endogenous nucleic acid sequence in a certain cell type. The 5 'or 3' fragment of nucleic acid non-coding for the region of the target gene is preferably placed in the vector according to its genomic arrangement at the 5 'end or the 3' end of the reporter gene. Any indicator gene known to a person skilled in the art whose expression can be detected in the cell can be used. A preferable used reporter gene is one which codes for chloramphenicol acetyl transferase (CAT), β-galactosidase (β-Gal) or lacZ. On the other hand, it is also possible to use a reporter gene that codes for a protein of interest, for example EPO, whose expression can be detected by immunological methods, for example ELISA. In a preferred embodiment, at least two vectors which contain different 5 'or / and 3' non-coding nucleic acid fragments of the target gene are each transfected into different cells and the expression of the reporter gene in the different cells is determined by methods known per se. a person familiar with the technique. It can be easily established with the process according to the invention which arrangement of the heterologous expression control sequence results in an optimal expression for a certain host cell. A further aspect of the invention relates to a process for providing a DHFR negative eukaryotic cell, preferably a mammalian cell and particularly a human cell, which is characterized in that: (a) the cell is transfected with a first vector comprising : (i) at least one target sequence for a site-specific recombinase, (ii) DNA sequences flanking the sequence (i) which are homologous to a nucleic acid sequence for DHFR that is endogenously present in the cell in order to allow homologous recombination and, (iii) optionally a positive selection marker gene and optionally a negative selection marker gene, (b) the transfected cell is cultured under conditions under which homologous recombination of the vector takes place, and (c) isolating the cell obtained according to step (b). In the process according to the invention, the recombinase target sequences and the homologous sequences are selected and used as explained in the above. The positive selection marker gene - if present - is placed between the sequences that are homologous to the DHFR gene. The negative selection marker gene - if present - is placed outside the homologous sequences. After homologous recombination has taken place at the DHFR locus, functional DHFR protein can not be synthesized by the cell. In this case, the vector sequences can be placed such that the promoter of the gene for DHFR is inactivated and / or so that a functional DHFR protein can no longer be synthesized due to an insertion or deletion in the coding sequence of the gene for DHFR. In order to inactivate both alleles of a gene for DHFR, the cells are first transfected with a vector according to the invention, and then selected and isolated. An allele of the gene for DHFR is inactivated by these cells, i.e. they are heterozygous (+/-) for the DHFR gene. These cells can then be transfected again with a vector according to the invention which preferably contains a positive selection marker gene that is different from the first vector. After the cells have been obtained in the selection stage in which both DHFR alleles are inactivated. Alternatively, an increase in the selection pressure can lead to a gene conversion and therefore to an inactivation of both alleles (see eg Mortensen et al., Mol.Cell. Biol. 12 (1992), 2391-2395) . The process according to the invention provides a negative DHFR cell whose use in a gene amplification process has the advantage that it does not synthesize an endogenous DHFR protein. When a selection step is carried out to amplify a heterologous nucleic acid sequence which is coupled to a nucleic acid sequence encoding a DHFR protein, the gene expression product for endogenous DHFR does not have an interference influence and therefore there is an increase in the efficiency of the gene amplification. Any suitable selectable marker gene leading to a selectable phenotype can be used as a positive selection marker gene, eg, antibiotic resistance. The nucleic acid sequence encoding the positive selection marker gene is preferably a gene that provides resistance to neomycin, kanamycin, geneticin or hygromycin.

Any negative selection marker gene known to a person familiar in the art may be used, the nucleic acid sequence encoding the negative selection marker gene is preferably a gene for thymidine kinase (TK) or / and the gene for hypoxanthine- guanine-phosphoribosyl transferase (HGPRT). The sequence flanked by the recombinase target sequences can be cut out of the cell genome by transient activation of the corresponding recombinase, for example, by: (a) transfecting the cell with a vector containing a nucleic acid sequence coding for a recombinase operably linked to an expression control sequence that is active in this cell, (b) culturing the transfected cell in this manner under conditions under which the recombinase is expressed and active, and (c) optionally isolating the cell. Not only is it possible with the processes according to the invention to inactivate a gene for DHFR, but also sequences of a gene for DHFR which are located between the sequences of the recombinase target gene as well as the selection marker gene introduced can be cut. of the genome of a cell by a recombinase-mediated reaction.

If the sequence flanked by the target recombinase sequences contains a positive selection marker gene, the cell containing this sequence is resistant to antibiotics. Therefore, it can be easily selected by methods known to a person familiar with the art. An additional advantage of the negative DHFR cell produced by the process according to the invention is that its properties can be characterized by methods known to a person familiar in the art and the cells can subsequently be used for other processes. In addition, the target sequence of recombinase introduced into the locus of the DHFR gene allows site-specific integration of nucleic acid sequences in the genome. A further preferred embodiment relates to a process for introducing a heterologous DHFR gene into a eukaryotic cell, which is characterized in that the negative DHFR cell obtained by one of the processes described above: (a) is transfected with a third vector that comprises: (i) optionally a positive selection marker gene which preferably differs from the negative selection marker gene of the first vector, (ii) a nucleic acid sequence encoding a DHFR, (iii) a nucleic acid sequence to be amplified encoding a protein in an expressible form in which each of the nucleic acid sequences of the partial sequences (i), (ii) and (iii) is flanked at the 5 'end and at the 3' end by at least one recombinase target sequence, (b) the transfected cell is cultured under conditions under which the acid sequence nucleic acid is flanked by recombinase target sequences and integrated into the recombinase target sequence that is already present in the genome of the cell, and (c) the cell obtained according to step (b) is isolated. The positive selection marker gene, the DHFR gene and the target gene encoding the desired protein are preferably operably linked each with an expression control sequence that is active or can be activated in the cell. In principle, a polyistronic construct with internal ribosomal binding sites is also possible. The nucleic acid sequence to be amplified by the target gene, however, is activated by a separate promoter. Particularly, the preferred expression control sequences are viral promoters / extenders. A CMV promoter is most preferred for the expression of the protein. It is advantageous to carry out the integration according to the invention of heterologous sequences in the genome of a cell in a site-specific manner and therefore to extrude the interferences in the heterologous sequences with genomic sequences. Therefore, this avoids the resulting disadvantages as described further above, such as clones of unstable production. In order to increase the expression rate of a heterologous nucleic acid sequence encoding a protein, it is possible to carry out an amplification step with methotrexate by known process steps. A further subject matter of the present invention is a vector comprising: (i) optionally a positive selection marker gene, (ii) a nucleic acid sequence encoding a DHFR, and (iii) a nucleic acid sequence in a expressible form coding for a desired protein in which each nucleic acid sequence of partial sequences (i), (ii), and (iii) is flanked at the 5 'end and at the 3' end by at least one sequence target of recombinase.

A further subject matter of the present invention is a vector for homologous recombination comprising: (i) optionally a positive selection marker gene, (ii) at least one target sequence for recombinase in each case whose flanks in the sequence (i) Y (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a nucleic acid sequence for DHFR that is endogenously present in a cell in order to allow homologous recombination and (iv) optionally a negative selection marker gene outside and preferably at the 3 'end of the homologous sequences (iii). In addition, the invention relates to a eukaryotic cell, preferably a human cell, obtainable by one of the processes described above. This cell is characterized in that: (a) at least one endogenous nucleic acid sequence encoding a DHFR is inactivated and preferably both endogenous alleles, (b) at least one target recombinase sequence is integrated into the genome in the region of this nucleic acid sequence encoding DHFR. Finally, a further subject matter of the invention is a eukaryotic cell, preferably a human cell, which is characterized by a heterologous nucleic acid sequence in the locus region of the endogenous DHFR gene, comprising: (i) an acid sequence nucleic acid encoding DHFR, (ii) a nucleic acid sequence encoding a desired protein, and (ii) at least one target recombinase sequence. The invention is illustrated by the following examples, the figures and the sequence protocol.

Legend of the figures Figure 1 (A) a vector for homologous recombination is shown which is used as the first vector. HR: homologous sequence, Sec 1: first heterologous expression control sequence, Rl: positive selection marker gene, loxP: loxP sequence with orientation, (B) shows the genomic sequences, (a) after completing the homologous recombination, ( b) after cutting a sequence flanked by loxP sequences catalyzed by a Cre recombinase, (C) shows a vector for integration mediated for Cre recombinase which contains a sequence placed between the loxP sequences (c) shows genomic sequences after the integration of a second vector in the loxP sequence, R2: positive selection marker gene, which optionally differs from Rl, Seq 2: second heterologous expression control sequence.

Figure 2 (A) shows a vector for homologous recombination HR: homologous sequence, R-box: positive and optionally negative selection marker gene, loxP: loxP sequence with orientation, HSV-tk: herpes simplex thymidine kinase; (B) shows a vector for homologous recombination with a homologous sequence on one side.

Figure 3 shows the expression of erythropoietin (EPO) controlled by the CMV / HIF promoter of HeLa S3 cells which were transfected with the vectors pHYG, pHIF-lc. and pARNT (pHIF-ljd) and whose EPO expression is measured in the supernatants of the cells 3, 4 and 5 days after the transfection. (erythropoietin concentration in μg / ml). pHYG: vector control, pHIF-lo-: a HIF-la cDNA under the control of a SRa promoter, pARNT: a HIF- / 3 cDNA under the control of a CMV promoter.

Figure 4 Four different vectors are shown in which each contains a CMV promoter (C) and the indicator gene / 3-galactosidase (B) in which non-coding nucleic acid fragments of the target gene (S) of different lengths have been inserted between these sequences. The length of the non-coding nucleic acid fragments is 0Kb in the A3-178 vector, 2.5 kb in the A3-177 vector, 3.7 kb in the A3-175 vector and 5.7 kb in the A3-131 vector. The control vector pNASS / 3 contains the 3-galactosidase reporter gene without a CMV promoter.

Figure 5 A measurement of the expression of the 3-galactosidase reporter gene is shown after transfection of HeLa S3 cells with the vectors of Figure 4 in a serial dilution (1: 2 to 128).

Figure 6 (A) shows the vector pNDI for homologous recombination at a locus of the DHFR gene. A positive selection marker gene (Neo) is flanked by two loxP sequences. The sequences that are homologous to a gene for DHFR (5 ', 3' DHFR region) are located at the 5 'end of one of the loxP sequences and on the 3' end of the other loxP sequence. (B) shows the vector pHDI for homologous recombination at a DHFR gene locus. A positive selection marker gene (Hyg) is flanked by two loxP sequences. Sequences that are homologous to a DHFR gene (5 ', 3' DHFR region) are located at the 5 'end of one of the loxP sequences and at the 3' end of the other loxP sequence.

Figure 7 (A) shows the genomic construction of a DHFR gene with an exon 1, exon 2 and exon 3, as well as the introns that are located between them, (B) shows a diagram of an objective construct corresponding to the vector of figure 6 , (C) shows the genomic structure after complete homologous recombination of the vector for homologous recombination in a DHFR gene. The distance between the EcoRI cleavage sites is 2.9 kb when the pNDI vector is used and 3.7 kb when the vector pHDI is used. Neo: neomycin, Hyg: hygromycin, kb: kilobases.

Figure 8 It shows a vector which contains a nucleic acid sequence encoding a protein X and a nucleic acid sequence encoding a DHFR protein which each includes regulatory sequences that are flanked by two loxP sequences. This vector can be used for Cre recombinase catalyzed integration within the genome in a loxP sequence.

The SEC. FROM IDE-S-T. NO.l shows a first nucleotide sequence of an HIF, SEC. OF IDET. NO .2 shows a second nucleotide sequence of HIF binding, SEC. FROM IDENT. NO.3 shows a loxP sequence EXAMPLES EXAMPLE 1 Expression of a gene for erythropoietin under the control of a CMV promoter and HIF overexpression The vectors pHYG, pHIF-la and pARNT (cf figure 3) are transfected in genetically modified HeLa S3 cells. A CMV promoter (cytomegalovirus) is introduced which controls EPO expression within the cells proximal to the translation start of the erythropoietin (EPO) gene of an EPO allele. The cells usually produce 1 μg of erythropoietin for 24 hours per 10 7 cells. 24 hours after transfection, they are passed at a concentration of 6 x 104 per 6-well plate. On the day of transfection the cells are incubated with a DNA-DOTAP mixture. The mixture contains 1.25 μg of the respective vector, 10 μl of DOTAP (Boehringer Mannheim 1202375) a final volume of 75 μl in 20 mM Hepes buffer per well. The mixture is preincubated for 10-15 minutes at room temperature. The cells are then incubated for 6 hours with the DNA-DOTAP in 3 ml of medium per well. Subsequently the cells are washed twice with PBS buffer and cultured in complete medium for 5 days. On day 3, 4 and 5, 100 μl of supernatant is removed each time and analyzed with ELISA for erythropoietin. The test is completed on day 5 and the cell count is determined. The amount of erythropoietin per well is calculated in relation to the same cell count (see figure 3). The examples show that an induction of the erythropoietin gene by HIF is still possible although a heterologous control sequence (CMV promoter) has been introduced into the promoter region of an allele of the erythropoietin gene. The measured increase in erythropoietin concentration indicates a synergistic effect of the factor induced by hypoxia or of the factors induced by hypoxia in both alleles. Therefore, it becomes apparent that the expression of an endogenous nucleic acid sequence can be increased by introducing a heterologous expression control sequence. If an activator (HIF) is expressed in the cell for which nucleic acid sequences are present in the expression control sequence, then expression of this gene can be further implemented. If corresponding sequences are not present at this locus of the gene, they can be introduced specifically into the genome by processes according to the invention, by means of homologous recombination.

Example 2 Optimized arrangement of an expression control sequence to increase the expression of an endogenous nucleic acid sequence The sequences at the 5 'end of an endogenous gene can stimulate expression and at the same time have repressor properties. When a heterologous expression control sequence is introduced into the genome at the 5 'end of the target gene, the level of expression is influenced by the 5' endogenous sequence. In order to obtain optimal expression of the target gene by means of a heterologous expression control sequence, it should be positioned so that the activity of the heterologous expression control sequence is not reduced by non-coding sequences at the terminus. 'of the target gene. A specific arrangement would be advantageous in order to provide synergistic effects of the individual sequence elements. In order to test the various arrangements of the heterologous expression control sequence, i.e., in order, for example, to determine at what distance the translation start of the coding sequence of the target gene should be integrated into the sequence of control of heterologous expression within the genome of the cell, different vectors are tested with different non-coding fragments of 5'-nucleic acid of the target gene (cf figure 4). The vectors described in figure 4 are transfected in HeLa S3 and the expression of the β-galactosidase reporter gene is measured (cf figure 5). 24 hours after the test the cells are passed at a concentration of 1 x 10 6 cells per 10 cm in petri dishes. On the day of transfection the cells are incubated with a DNA-DOTAP mixture. The mixture contains 1 pmol of the respective vector, (A3-178, A3-177, A3-175, A3-181 or pNASS / S, see figure 4) in 60 μl of DOTAP (Boehringer Mannheim 1202375) constituted up to 300 μl with 20 mM HEPES buffer solution. The mixture is incubated for 10-15 minutes at room temperature. The cells are preincubated for 6 hours with DOTAP-DNA in 6 ml of serum-free medium per petri dish. Subsequently the cells are washed twice with PBS buffer and cultured in complete medium for 22 hours. In order to measure the expression of jß-galactosidase, the cells are isolated in 200 μl of PBS and lysed by freezing at -20 ° C and reheating. 10 μl of the 1:10 lysate is diluted with substrate (3.29 mM-β-D-galactopyranoside chlorophenol red (Boehringer Mannheim 884308), 100 mM HEPES, 150 M NaCl, 22 mM MgCl, 1% BSA, Triton-X 100 al 0.1%, 1% sodium azide, pH 7. The samples are diluted in stages 1: 2 and incubated at 37 ° C in a 96-well plate until a dark red color is formed. 570/580 nm, or 550 nm As shown in Figure 5, expression of the reporter gene is highest in cells that have been transfected with the vector A3-178.In this vector, the control sequence of heterologous expression is proximal to the translation start of the coding sequence.

Therefore, this method can be used to determine in a simple and rapid manner which arrangement of a control sequence of heterologous expression in the genome of a host cell should be selected in order to obtain optimal expression of an endogenous target gene.

Example 3 Production of negative DHFR cells In a first step, the vectors for recombination according to the invention are prepared. These vectors are transfected into human cell lines in a second stage and analyzed for homologous recombination events. In this way, one can be inactivated first and then the second allele for the DHFR gene.

DHFR vector for homologous recombination The gene for human DHFR is located on chromosome 5 and comprises 30 kb which are placed in 6 exons. A 1.8 kb EcoRI fragment containing parts of the promoter, parts of exon 2 and complete exon 1 is used to prepare the vector for homologous recombination. Exon 1 is removed by an AapI digestion and the Neo gene (1.4 kb) or Hyg resistance (2.2 kb) is inserted into the resulting space (0.45 kb) by means of linkers. These linkers contain the minimum sequence TAT TG AAG CAT ATT ACÁ TAC GAT ATG CTT CAA TA (loxP sequence) in addition to the adapter nucleotides. The linker sequences are placed in the same orientation and the resistance gene preferably is in an antisense orientation relative to the DHFR gene. After the resistance gene has been inserted, the region of homology is enlarged. For this, the vector is ignited by EcoRI fragment from the 3 'region (6.0 kb) (Figure 6). In this way, one obtains the target constructs pNDI (11.5 kb) and pHDI (12.3 kb) according to the invention. After the homologous recombination has been completed, complete exon 1 (amino acids 1-28) and parts of the gene promoter for DHFR have been removed. The cell can no longer express a functional DHFR protein.

Transfection of cells The human cell lines used can not be polyploid for chromosome 5 and have not been kept under selection by MTX. In both cases, more than 2 alleles should have been inactivated.

HeLa S3 cells (ATCC CCL-2.2) Cells are grown in tissue culture flasks in RPMI 1640 medium, 10% fetal bovine serum, 2 mM L-glutamine and 1 mM MEM (non-essential amino acids). Incubation is carried out at 37 ° C and 5% C02. The electroporation buffer contains 20 mM Hepes, 138 mM NaCl, 5 mM KCl, 0.7 mM Na2HP04 and 6 mM D-glucose monohydrate, pH 7.0. 10 μg linearized vector DNA (pNDI) (Biorad Gene Pulser) is electroporated in 1 x 107 cells at 960 μF and 250V. After electroporation the cells are taken up in medium containing 600 μg / ml G418 (geneticin, Boehringer Mannheim) and cultured. After 10 days of selection (the medium is changed every two days) the positive clones are isolated and expanded. HT1080 cells (ATCC CCL-121) The cells are cultured and selected as described for HeLa S3 cells using DMEM medium containing 10% fetal bovine serum, 2 mM L-glutamine and 1 mM sodium pyruvate.

Namalwa cells (ATCC CRL-1432) This cell line is a cell line in suspension and should be cultured accordingly. The medium corresponds to that described for Hela S3 cells. After transfection, the cells are distributed among 40 wells in a 96-well plate. The positive clones are expanded in wells in 48-, 24-, 12- and 6-well plates. The selection is carried out in 1 mg / ml of G418.

Detection of negative DHFR cells (+/-) Insertion of the vector is detected by means of Southern blot analysis or PCR. If homologous recombination has occurred correctly, a band of 2.9 kb is detected after digestion with EcoRI which has been done by inserting the Neo gene in addition to a 1.8 kb band which represents the intact DHFR gene (figure 7c) . The mixed clones (unequal ratio of band intensities in Southern blotting) are separated by single cell deposition in a FACS, subcloned and subsequently expanded. An allele of the gene for DHFR is inactivated in clones that have been identified as positive.

Production of DHFR-negative cells (- / -) Clones of cells in which the DHFR allele is inactivated (+/-) can undergo a renewed homologous recombination. For this, they are transferred as described above with 10 μg of linearized DNA from the pHDI vector. The selection is carried out in a medium containing 500 μg / ml hygromycin B (Boehringer Mannheim). Increasing the concentration of G418 in the medium increases the selection pressure on DHFR +/- cells and DHFR - / - cells are obtained. A genetic conversion leads to an interchromosomal recombination which is the way in which the second DHFR allele is inactivated. The DHFR - / - cells contain two inactivated DHFR alleles and can no longer synthesize tetrahydrofolate. Therefore, thymidine, glycine and purine should be added to the medium (supplementation) Optionally, the cells are grown in medium a 'cells (Gibco BRL). The DHFR - / - cells are detected as described above. A wild-type band (1.8 kb) is detectable in homozygous negative DHFR cells. Cells that have been transfected with pDHI show a new 3.7 kb band in the Southern EcoRI blot after homologous recombination (Figure 7c).

Use of negative DHFR cells (- / -) The cells according to the invention can be used for large-scale production of proteins. For this, a vector according to the invention (according to Figure 8) and an expression vector encoding a Cre recombinase are transfected into DHFR - / - cells. Cre recombinase removes antibiotic resistance from the gene locus for DHFR and inactivates the vector according to the invention in the loxP sequence in the cell genome DHFR - / -. The cells are again sensitive to antibiotic and independent of thymidine, glycine and purine supplementation. The selection can be carried out by using a medium without supplementation or by adding a suitable antibiotic to the culture medium. In this case, the antibiotic corresponds to the resistance gene which has been removed by the Cre recombinase of the cell genome. If the vector integrated in the loxP sequence contains a positive selection marker gene, the selection can be carried out by adding this antibiotic to the medium.

Increase of the yield of production by amplification of genes In order to increase the production yield of the cells for the recombinant protein, a selection is made by methotrexate (MTX) which amplifies the gene for DHFR introduced into the cell and the heterologous nucleic acid sequence coding for a protein. In order to obtain an amplification, the cells are grown together in the presence of increasing concentrations (100-1000 mM) of MTX. The degree of amplification is monitored by comparative Southern blotting cytometric evaluation (before, during and after the addition of MTX). The cells according to the invention obtained after the amplification step contain many copies of the gene introduced for DHFR and of the heterologous nucleic acid sequence inserted in the loxP locus. They are characterized by a high production yield of the heterologous nucleic acid. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following: CLAIMS 1. A process for changing the expression of a nucleic acid sequence which is endogenously present in a eukaryotic cell, the process is characterized in that (a) the cell is transfected with a first vector comprising: (i) at least one sequence that is selected from a first heterologous expression control sequence and a first amplification gene, (ii) a positive selection marker gene , (iii) at least two target sequences for a site-specific recombinase, flanking sequences (i) and (ii), (iv) DNA sequences which flank sequences (i), (ii) and ( iii) and that are homologous to the nucleic acid section in the genome of the cell in order to allow homologous recombination, (b) the transfected cell is cultured under conditions which homologous recombination of the vector takes place, and (c) ) the cell obtained according to step (b) is isolated. 2. The process according to claim 1, characterized in that loxP sequences are used as target recombinase sequences. 3. The process according to claim 1 or 2, characterized in that the cell is a human cell. 4. The process according to one of the preceding claims, characterized in that the cell is an immortalized cell. 5. The process according to claim 4, characterized in that the cell is an HT1080, Namalwa or HeLa S3 cell. The process according to one of the preceding claims, characterized in that the heterologous expression control sequence contains a promoter / extender, preferably a viral promoter and particularly preferable a CMV promoter. The process according to one of claims 1 to 6, characterized in that the heterologous expression control sequence contains a 3 'non-coding sequence. The process according to one of the preceding claims, characterized in that the homologous sequences are selected such that an endogenous expression control sequence of the nucleic acid sequence that is endogenously present is removed by homologous recombination. 9. The process according to one of the preceding claims, characterized in that the positive selection marker gene is a gene that provides resistance to neomycin, kanamycin, geneticin or hygromycin. The process according to one of the preceding claims, characterized in that the vector additionally contains a negative selection marker gene which is placed outside in the homologous sequences, according to claim 1 (a) - (iv). The process according to one of the preceding claims, characterized in that the nucleic acid sequence that is located between the recombinase target sequences is cut from the cell genome by transient activation of a site-specific recombinase that recognizes the target sequences . The process according to claim 1, characterized in that: (a) the cell is transfected with an additional vector, comprising: (i) at least one sequence that is selected from a second control sequence of heterologous expression and a second amplification gene, (ii) a positive selection marker gene which preferably differs from the positive selection marker gene of the first vector, and (iii) at least two recombinase target sequences flanking sequences (i) and ( ii) (b) the transfected cell is cultured under conditions under which the sequence flanked by the target sequences is integrated into the target sequence in the genome of the cell, (c) the cell obtained according to the step is isolated ( b), and (d) steps (a) to (c) are optionally repeated at least once with expression control sequences or / and amplification genes which vary, in each case. 13. A vector for homologous recombination, characterized in that it comprises: (i) at least one sequence that is selected from an expression control sequence and an amplification gene, (ii) a positive selection marker gene, (iii) by at least two target sequences for a site-specific recombinase which flank sequences (i), and (ii), (iv) DNA sequences flanking sequences (i), (ii) and (iii) which they are homologous to a nucleic acid section in the genome of a cell in order to allow homologous recombination, and (v) optionally a negative selection marker gene. 14. A vector, characterized in that it comprises: (i) at least one sequence that is selected from a heterologous expression control sequence and an amplification gene, (ii) a positive selection marker gene, (iii) at least two recombinase target sequences which flank sequences (i) and (ii), (iv) optionally a negative selection marker gene. 15. A eukaryotic cell, preferably a human cell, characterized in that it can be obtained by the process according to one of claims 1 to 12. 16. A eukaryotic cell, preferably a human cell characterized in that: (a) it contains at least a chromosomally located sequence, which is selected from a heterologous expression control sequence and an amplification gene in operable linkage with a nucleic acid sequence that is endogenously present and, (b) this sequence is flanked by recombinase target sequences. 17. A process for changing the expression of a nucleic acid sequence that is endogenously present in a eukaryotic cell, characterized in that it comprises: (a) the cell is transfected by a vector comprising: (i) at least one nucleic acid sequence that it binds to an activating protein, (ii) a positive selection marker gene, (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a nucleic acid section in the genome of the cell in order to allow homologous recombination, (b) the transfected cell is cultured under conditions under which homologous recombination of the vector takes place, and (c) the cell obtained according to step (b) is isolated. 18. The process according to claim 17, characterized in that at least one nucleic acid sequence is used that binds to the hypoxia inducible factor (HIF). 19. The process according to claim 18, characterized in that the nucleic acid sequence that binds HIF is selected from the 53 bp sequence, in accordance with SEQ. DE IDE T. NO.l, of the 43 bp sequence in accordance with SEC. FROM IDENT. NO.2, a sequence that is homologous to these sequences or a sequence which hybridizes with these sequences under restriction conditions. The process according to one of claims 17 to 19, characterized in that it additionally comprises transfecting the cell with a vector comprising: (i) a nucleic acid sequence encoding an activating protein which is operably linked to a sequence of active expression control in this cell, and (ii) optionally a positive selection marker gene. 21. The process according to claim 20, characterized in that the activator protein is a HIF-lc protein. or / and a HIF-1/3 protein. 22. The process according to one of claims 18 to 21, characterized in that the cells are cultured at a concentration of 02 of 0.1 to 2%. 23. A vector for homologous recombination, characterized in that it comprises: (i) at least one nucleic acid sequence which binds to an activating protein, (ii) a positive selection marker gene, (iii) flanking DNA sequences to sequences (i) and (ii) which are homologous to a section of nucleic acid in the genome of the cell in order to allow homologous recombination. 24. A eukaryotic cell, preferably a human cell, characterized in that it can be obtained by a process according to one of claims 17 to 21. 25. A eukaryotic cell, preferably a human cell, characterized in that it contains at least one fragment of chromosomally located heterologous nucleic acid, which binds to an activating protein / activator protein complex which is operably linked to a gene that is endogenously present in the cell. 26. A process for testing the influence of non-coding nucleic acid sequences from the region of a target gene endogenously present in a eukaryotic cell on its expression, the process is characterized in that: (a) the cell is transfected by a vector comprising : (i) a heterologous expression sequence that is active or that can be activated in the cell, which is operably linked to a reporter gene, and (ii) non-coding nucleic acid fragments at the 5'-end and / or the 3 'end of the target gene region, (b) the cell is cultured under conditions under which the expression control sequence is activated, and (c) expression of the reporter gene is measured. 27. The process according to claim 26, characterized in that the reporter gene codes for chloramphenicol acetyltransferase (CAT), β-galactosidase (/ S-Gal) or lacZ. The process according to one of claims 26 or 27, characterized in that: (a) at least two vectors which contain non-coding fragments of 5 'or 3' nucleic acid of the target gene that are different from each other, they are transfected into different cells in each case, and (b) the expression of the reporter gene in the different cells is determined. 29. A process for providing a DHFR negative eukaryotic cell, characterized in that: (a) the cell is transfected with first vector comprising: (i) at least one target sequence for a site-specific recombinase, (ii) DNA sequences flanking the sequence (i) which are homologous to a nucleic acid sequence for DHFR that is present endogenously in the cell in order to allow homologous recombination, and (iii) optionally a positive selection marker gene and optionally a Negative selection marker gene, (b) the transfected cell is cultured under conditions under which homologous recombination of the vector takes place, and (c) the cell obtained according to step (b) is isolated. 30. The process according to claim 29, characterized in that loxP sequences are used as the target recombinase sequences. 31. The process according to one of claims 29 or 30, characterized in that the nucleic acid sequence encoding the positive selection marker gene is a gene that provides resistance to neomycin, kanamycin, geneticin or hygromycin. 32. The process according to one of claims 29 to 31, characterized in that the nucleic acid sequence encoding the negative selection marker gene is a thymidine kinase (TK) gene or / and a gene for hypoxanthine-guanine- phosphoribosyltransferase. 33. The process according to one of claims 29 to 32, characterized in that the sequence that is flanked by the target recombinase sequences is cut from the genome of the cell by transient activation of the corresponding recombinase. 34. A process for introducing a heterologous DHFR gene into a eukaryotic cell, the process is characterized in that it comprises: a cell obtained by the process according to claim 33, which: (a) is transfected with a third vector that comprises: (i) optionally a positive selection marker gene which preferably differs from the positive selection marker gene of the first vector, (ii) a nucleic acid sequence encoding DHFR, (iii) a nucleic acid sequence to be amplified , which codes for a protein in which each nucleic acid sequence of the partial sequences (i), (ii) and (iii) at the 5 'end and at the 3' end by at least one target recombinase sequence, (b) the transfected cell is cultured under conditions under which the nucleic acid sequence flanked by target sequences of The recombinase is integrated into the target sequence of the recombinase that is already present in the genome of the cell, and (c) the cell obtained according to step (b) is isolated. 35. A vector, characterized in that it comprises: (i) optionally a positive selection marker gene, (ii) a nucleic acid sequence encoding a DHFR, and (iii) a nucleic acid sequence in an expressible form coding for a desired protein, in which each nucleic acid sequence of the partial sequences (i), (ii) and (iii) is flanked at the 5 'end and at the 3' end by at least one target recombinase sequence. 36. A vector for homologous recombination, characterized in that it comprises: (i) optionally a positive selection marker gene, (ii) at least one target recombinase sequence in each case which flanks sequence (i), (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a nucleic acid sequence for DHFR that is endogenously present in a cell in order to allow homologous recombination, and (iv) optionally a negative selection marker gene outside the homologous sequences (iii). 37. A eukaryotic cell, preferably a human cell, characterized in that it can be obtained by a process according to one of claims 29 to 34. 38. A eukaryotic cell, preferably a human cell, characterized in that: (a) at least an endogenous nucleic acid sequence encoding a DHFR is inactivated, and (b) at least one target recombinase sequence is integrated into the genome in the region of this nucleic acid sequence encoding DHFR. 39. A eukaryotic cell, preferably a human cell, characterized in that it has a heterologous nucleic acid sequence in the region of a locus of the endogenous gene for DHFR, comprising: (i) a nucleic acid sequence encoding DHFR, (ii) ) a nucleic acid sequence encoding a desired protein, and (iii) at least one target recombinase sequence.

SEQUENCE PROTOCOL (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Boehringer Mannheim GmbH (B) PATH: Sandhofer Strasse 112-132 (C) CITY: Mannheim (E) COUNTRY: Germany (F) POSTAL CODE: D-68305 (ii) TITLE OF THE INVENTION: Optimization of cells for endogenous activation of genes (iii) SEQUENCE NUMBER: 3 (iv) READABLE COMPUTER FORM: (A): Flexible disk (B) COMPUTER: IBM Compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE OR PROGRAM: Patentln Relay '1.0, version # 1.30 (EPO) (2) INFORMATION FOR SEC. FROM IDENT. NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: both (D) TOPOLOGY: linear (ix) DESCRIPTION OF THE SCUENCY: SEC. FROM IDENT. NO: 1: CCTCTCCTCT AGGCCCGTGG GGCTGGCCCT GCACCGCCGA CTTCCCGGG ATG 53 (2) INFORMATION FOR SEC. FROM IDENT. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 43 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: both (D) TOPOLOGY: linear (ix) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2 CTACGTGCTG TCTCACACAG CCTGTCTGAC CTCTCGACCC TAC 43 (2) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: both (D) TOPOLOGY: linear (ix) DESCRIPTION OF THE SEQUENCE: SEC. ID: 3 TATTGAAGCA TATTACATAC GATATGCTTC AATA 34

Claims (19)

1. A process for changing the expression of a nucleic acid sequence which is endogenously present in a eukaryotic cell, the process is characterized in that (a) the cell is transfected with a first vector comprising: (i) at least one sequence that is selected from a first heterologous expression control sequence and a first amplification gene, (ii) a positive selection marker gene , (iii) at least two target sequences for a site-specific recombinase, flanking sequences (i) and (ii), (iv) DNA sequences which flank sequences (i), (ii) and ( iii) and that are homologous to the nucleic acid section in the genome of the cell in order to allow homologous recombination, (b) the transfected cell is cultured under conditions which homologous recombination of the vector takes place, and (c) ) the cell obtained according to step (b) is isolated.

2. The process according to claim 1, characterized in that loxP sequences are used as target recombinase sequences.

3. The process according to one of the preceding claims, characterized in that the vector additionally contains a negative selection marker gene which is placed outside the homologous sequences according to claim 1 (a) (iv). The process according to one of the previous claims, characterized in that one of the nucleic acid sequences is located between the recombinase target sequences and is cut in the genome of the cell by transient activation and a site-specific recombinase that recognizes the objective sequences. 5. A vector for homologous recombination, characterized in that it comprises: (i) at least one sequence that is selected from an expression control sequence and an amplification gene, (ii) a positive selection marker gene, (iii) by at least two target sequences for a site-specific recombinase which flank sequences (i), and (ii), (iv) DNA sequences flanking sequences (i), (ii) and (iii) which are homologous to a section of nucleic acid in the genome of a cell in order to allow homologous recombination, and (v) optionally a negative selection marker gene. 6. A vector, characterized in that it comprises: (i) at least one sequence that is selected from a heterologous expression control sequence and an amplification gene, (ii) a positive selection marker gene, (iii) at least two recombinase target sequences which flank sequences (i) and (ii) (iv) optionally a negative selection marker gene. A eukaryotic cell, preferably a human cell, characterized in that: (a) it contains at least one chromosomally located sequence that is selected from a heterologous expression control sequence and an amplification gene in operable binding with a nucleic acid sequence which is present endogenously and (b) this sequence is flanked by recombinase target sequences. 8. A process for changing the expression of a nucleic acid sequence that is present endogenously in a eukaryotic cell, characterized in that it comprises: (a) the cell is transfected by a vector comprising: (i) at least one acid sequence nucleic that binds to an activating protein, (ii) a positive selection marker gene, (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a section of nucleic acid in the genome of the cell in order to allow homologous recombination, (b) the transfected cell is cultured under conditions under which homologous recombination of the vector takes place, and (c) the cell obtained in accordance with step (b) is isolated . 9. The process according to claim 8, characterized in that at least one nucleic acid sequence is used that binds to the hypoxia inducible factor (HIF). 10. A vector for homologous recombination, characterized in that it comprises: (i) at least one nucleic acid sequence which binds to an activating protein, (ii) a positive selection marker gene, (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a section of nucleic acid in the genome of the cell in order to allow homologous recombination. 11. A eukaryotic cell, preferably a human cell, characterized in that it can be obtained by a process according to one of claims 8 to 10. 12. A eukaryotic cell, preferably a human cell, characterized in that it contains at least one fragment of chromosomally located heterologous nucleic acid, which binds to an activating protein / activator protein complex which is operably linked to a gene that is endogenously present in the cell. 13. A process for testing the influence of non-coding nucleic acid sequences from the region of a target gene endogenously present in a eukaryotic cell on its expression, the process is characterized in that: (a) the cell is transfected by a vector comprising (i) a heterologous expression control sequence that is active or that can be activated in the cell, which is operably linked to a reporter gene, and (ii) non-coding nucleic acid fragments at the 5 'end or / and at the 3 'end of the target gene region, (b) the cell is cultured under conditions under which the expression control sequence is activated, and (c) expression of the reporter gene is measured. 1

4. A process for providing a DHFR negative eukaryotic cell, characterized in that: (a) the cell is transfected with first vector comprising: (i) at least one target sequence for a site-specific recombinase, (ii) DNA sequences flanking the sequence (i) which are homologous to a nucleic acid sequence for DHFR that is present endogenously in the cell in order to allow homologous recombination, and (iii) optionally a positive selection marker gene and optionally a negative selection marker gene, (b) the transfected cell is cultured under conditions under which homologous recombination of the vector takes place, and (c) the cell obtained according to step (b) is isolated. 1

5. A process for introducing a heterologous DHFR gene into a eukaryotic cell, the process is characterized in that it comprises: a cell obtained by the process according to claim 14, which: (a) is transfected with a third vector that comprises: (i) optionally a positive selection marker gene which preferably differs from the positive selection marker gene of the first vector, (ii) a nucleic acid sequence encoding DHFR, (iii) a nucleic acid sequence to be amplified , which encodes a protein in an expressible form in which each nucleic acid sequence of the partial sequences (i), (ii) and (iii) at the 5 'end and at the 3' end by at least one target sequence of recombinase, (b) the transfected cell is cultured under conditions under which the nucleic acid sequence flanked by recombinase target sequences is integrated into the target sequence of the e recombinase that is already present in the genome of the cell, and (c) the cell obtained according to step (b) is isolated. 1

6. A vector, characterized in that it comprises: (i) optionally a positive selection marker gene, (ii) a nucleic acid sequence encoding a DHFR, and (iii) a nucleic acid sequence in an expressible form coding for a desired protein, in which each nucleic acid sequence of the partial sequences (i), (ii) and (iii) is flanked at the 5 'end and at the 3' end by at least one target recombinase sequence. 1

7. A vector for homologous recombination, characterized in that it comprises: (i) optionally a positive selection marker gene, (ii) at least one target recombinase sequence in each case which flanks sequence (i), (iii) DNA sequences flanking sequences (i) and (ii) which are homologous to a nucleic acid sequence for DHFR that is endogenously present in a cell in order to allow homologous recombination, and (iv) optionally a negative selection marker gene outside the homologous sequences (iii). 1

8. A eukaryotic cell, preferably a human cell, characterized in that: (a) at least one endogenous nucleic acid sequence encoding a DHFR is inactivated, and (b) at least one target recombinase sequence is integrated into the genome in the region of this nucleic acid sequence that codes for DHFR. 1

9. A eukaryotic cell, preferably a human cell, characterized in that it has a heterologous nucleic acid sequence in the region of an endogenous gene locus for DHFR, comprising: (i) a nucleic acid sequence encoding DHFR, (ii) ) a nucleic acid sequence encoding a desired protein, and (iii) at least one target recombinase sequence.