HK1081998B - Sequence specific dna recombination in eukaryotic cells - Google Patents

Description

Sequence-specific DNA recombination in eukaryotic cells

The present invention relates to a method for sequence-specific recombination of DNA in eukaryotic cells, comprising introducing a first DNA comprising a nucleotide sequence comprising at least one recombination sequence into a cell, introducing a second DNA comprising a nucleotide sequence comprising at least one further recombination sequence into a cell, and performing sequence-specific recombination by a bacteriophage lambda integrase Int.

The controlled manipulation of eukaryotic genomes and the expression of recombinant proteins from episomal vectors are important methods for analyzing the function of specific genes in living organisms. Furthermore, the manipulation also plays a role in a gene therapy method in medicine. In this context, the production of transgenic animals, the alteration of genes or gene fragments (so-called "gene targeting"), and the targeted integration of foreign DNA into the genome of higher eukaryotes are of particular importance. Currently, these techniques can be improved by the identification and application of sequence-specific recombination systems.

In addition, the introduction of expression cassettes which encode and express the desired polypeptides/products into the genome of biotechnologically relevant host cells via sequence-specific integration is also of greater importance for the production of biopharmaceuticals. In stably transformed cell lines, the level of expression of the desired polypeptide depends on the site of integration. By sequence-specific integration, sites with high transcriptional activity can be preferentially used. Conventional methods for generating production cell lines expressing a desired polypeptide/product are based on the random integration of a recombinant expression vector into the genome of a host cell. In stably transformed cell lines, changes in the expression level of the integrated gene of interest are primarily due to differences in chromosomal location and copy number. Random integration of adjacent heterochromatin results in variable transgene expression levels. Chromosomal locations that promote expression of integrated target genes are considered transcriptionally active regions of euchromatin. This random nature of integration allows for a great diversity in recombinant cell durability, productivity, and quality, necessitating a sophisticated screening method to identify and isolate suitable cell clones expressing high levels of the desired polypeptide. Furthermore, heterogeneity also means that an optimized production method has to be developed for each clone, which makes the development of suitable production cell lines a time-consuming, labor-intensive and expensive process.

Conserved sequence-specific DNA recombinases fall into two families. The first family, members of the so-called "integrase" family, catalyses the breaking and rejoining of DNA strands between two defined nucleotide sequences (hereinafter referred to as recombination sequences). The recombination sequences are either on two different DNA molecules or on one DNA molecule, resulting in intermolecular or intramolecular recombination, respectively. For intramolecular recombination, the reaction results depend on the respective orientation of the recombination sequences to each other. In the case of inversion, i.e.in the opposite direction of the recombination sequences, the DNA segments between the recombination sequences are inverted. In the case of direct orientation, i.e.where the recombination sequences repeat in tandem on the DNA substrate, deletions occur. In the case of intermolecular recombination, i.e., if two recombination sequences are located on two different DNA molecules, the two DNA molecules may be fused. Members of the integrase family often catalyze both intramolecular and intermolecular recombination, while recombinases of the second family, the so-called "invertases/resolvases", can only catalyze intramolecular recombination.

Currently, recombinases used to manipulate eukaryotic genomes belong to the integrase family. The recombinases are the Cre recombinase of the P1 phage and the Flp recombinase from yeast (Muller, U. (1999) Mech. Develop., 82, pp.3). The recombinant sequence to which Cre recombinase binds is designated loxP. LoxP is a 34bp long nucleotide sequence consisting of two 13bp long inverted nucleotide sequences and an 8bp long spacer located between the inverted sequences (Hoess, r.et al (1985) j.mol.biol., 181, pp.351). The Flp binding sequence designated FRT has a similar structure. However, they differ from loxP (Kilby, j.et al (1993) Trends genet, 9, pp.413). Thus, the recombination sequences cannot be substituted for each other, i.e., Cre cannot recombine the FRT sequence and FLP cannot recombine the loxP sequence. Both recombination systems are active for long distances, i.e. the DNA fragment that is inverted or deleted and flanked by two loxP or FRT sequences may be several 10000 base pairs long.

For example, the two systems can be used to achieve tissue-specific recombination in mouse systems, chromosomal translocation in plants and animals, and controlled induction of gene expression; a review article by muller, u. (1999) mech. devilop., 82, pp.3. In a specific tissue of the mouse, the DNA polymerase β is deleted in this way; gu, H.et al (1994) Science, 265, pp.103. As another example, DNA tumor virus SV40 oncogene is specifically activated in mouse lenses, resulting in exclusive tumor formation in these tissues. The Cre-loxP strategy can also be used in combination with inducible promoters. For example, expression of the recombinase is regulated by an interferon-inducible promoter, resulting in deletion of a particular gene in the liver but not (or only to a low degree) in other tissues; kuhn, R.et al (1995) Science, 269, pp.1427.

To date, 3 members of the invertase/resolvase family have been used to manipulate eukaryotic genomes. The mutant of Mu phage invertase Gin catalyzes the inversion of DNA fragments in plant protoplasts without the need for cofactors (cofactors). However, it has been found that this mutant has a superior capacity to cause recombination, i.e. it also catalyzes the breaking of DNA strands at other positions than its native recombination sequence. This results in an undesirable partial lethal recombination event in the plant protoplast genome. Beta-recombinases from streptococcus pyogenes (streptococcus pyogenes) catalyze recombination between two recombination sequences as direct repeats in mouse cell cultures, resulting in fragment deletions. However, inversions were also detected at the same time as the deletion, which makes this system unsuitable for controlled use in the manipulation of eukaryotic genomes. Mutants of the E.coli gamma delta resolvase have been shown to be active against episomal recombination sequences and artificially introduced genomic recombination sequences, but the efficiency of the latter reaction is still rather low.

Manipulation of eukaryotic genomes with Cre and Flp recombinase, respectively, shows significant disadvantages. In case of deletion, i.e. recombination of two tandem repeats of loxP or FRT recombination sequences in the genome, there is an irreversible loss of DNA fragments between said tandem repeats. Thus, the gene located in the DNA fragment is permanently lost from the cell and organism. Therefore, it is not possible to reconstruct the initial state in order to carry out a new analysis of the function of the genes, for example at a later developmental stage of the organism. Irreversible loss of a DNA fragment by deletion can be avoided by inversion of the corresponding DNA fragment. The gene can be inactivated without being lost by inversion, and then can be reactivated by timely regulation of recombinase expression by back recombination (back recombination) in later developmental stages or adult animals. However, the use of Cre and Flp recombinases in this improved method has the following disadvantages: the inversion cannot be regulated because the recombination sequence is not altered by the recombination event. Thus, recombination events occur repeatedly, causing the inactivation of the respective genes, since the inversion of the corresponding DNA fragments reaches the reaction equilibrium only in some target cells, up to 50% of the target cells. Attempts have been made to solve, at least partially, this problem by constructing mutated loxP sequences which cannot be used for further reactions after one recombination. However, this disadvantage is characteristic of the reaction, i.e.after inactivation of the gene by inversion, the gene cannot be subsequently activated by recombination in return.

Another disadvantage of the Flp recombinase is its reduced thermostability at 37 ℃ which significantly limits the efficiency of the recombination reaction in higher eukaryotes, e.g.mice with a body temperature of about 39 ℃. Thus, a Flp mutant with higher thermostability than the wild type recombinase was prepared. However, even these mutated Flp enzymes still show lower recombination efficiency than Cre recombinase.

Another use of sequence-specific recombinases is in the medical field, e.g. gene therapy, where the recombinase integrates a desired DNA segment into the genome of a corresponding human target cell in a stable and controlled manner. Both Cre and Flp catalyze intermolecular recombination. These two recombinases recombine plasmid DNA with one copy of its respective recombination sequence with the corresponding recombination sequence that has been previously inserted into the eukaryotic genome via homologous recombination. However, it is expected that this reaction will involve recombination sequences that are "naturally" occurring in the eukaryotic genome. Because loxP and FRT are 34 and 54 nucleotides long, respectively, it is statistically unlikely that these recombination sequences that are part of the genome will be exactly matched. Even in the presence of recombination sequences, the disadvantage of the above-mentioned reversion reaction still exists, i.e.both Cre and Flp recombinases can excise the inserted DNA fragment after successful integration by intramolecular recombination.

It is therefore a problem of the present invention to provide a simple and controllable reconstitution system and the required method of operation (working means). Another problem of the present invention is to provide a recombination system and a desired method of operation which allow stable targeted integration of a desired DNA sequence. It is a further problem of the present invention to provide a method which allows the production of improved protein expression systems based on one of these recombinant systems.

The problem is solved by the subject matter described in the claims.

The present invention is explained in more detail by the following examples.

FIG. 1 is a schematic representation of the recombination reaction, i.e.the integration and excision catalyzed by the wild-type integrase Int. The supercoiled plasmid DNA with one copy of the recombination sequence attP is shown (top). AttP contains 5 so-called Int arm binding sites (P1, P2, P1 ', P2 ', P3 '), 2 core Int binding sites (C and C '; indicated by black arrows), 3 IHF binding sites (H1, H2, H '), 2 Xis binding sites (X1, X2) and so-called overlap regions (open rectangles) where the actual DNA strand exchange takes place. The natural pairing (partner) sequence of attP, attB, containing 2 Int core binding sites (B and B'; indicated by open arrows) and overlapping regions is shown on the linear DNA below. For recombination between attB and attP, Int and IHF are necessary for integration of the plasmid into the DNA fragment with attB. Thereby forming two new hybrid recombination sequences attL and attR as target sequences for excision. The latter reaction requires wild-type Int and IHF, and another cofactor XIS encoded by the lambda phage.

FIG. 2 shows intramolecular and intermolecular recombination reactions. (A) Is an intramolecular integration (attB × attP) recombination. (B) Is an intermolecular integration (attB × attP) recombination. (C) Is intramolecular excision (attL × attR) recombination. (D) Is intermolecular excision (attL × attR) recombination. The substrate vectors and the expected recombinant products are drawn schematically at the top of each group. The proportion of GFP-expressing cells was measured by FACS at 3 time points after co-transfection with substrate and expression vector. We show the mean of the triplicate analyses and the standard deviation represented by the vertical line.

FIG. 3 shows that the presence of the Int arm-binding DNA sequence at the att site stimulates intermolecular recombination. (A) Are pairs of intermolecular recombination substrate vectors containing attB or attP in different combinations, and the resulting product driven by the CMV promoter to express GFP. (B) Various combinations of substrate vectors are co-transfected with expression vectors for wild-type Int, mutant Int-h or Int-h/218. Cells were analyzed by FACS for 48h and the ratio of GFP-expressing cells for both pairs of substrates was measured. As shown, reference is made to recombination between attP and attP. We show the mean of the triplicate analyses and the standard deviation represented by the vertical line. Actual average values (%) of Int GFP-expressing cells were 0.08 (B.times.B), 1.24 (P.times.P), and 0.81 (P.times.B). Int-h is 1.15 (B.times.B), 8.07 (P.times.P), and 9.90 (P.times.B). Int-h/218 is 4.01 (B.times.B), 17.62 (P.times.P), and 16.45 (P.times.B).

FIG. 4 shows the stimulation of both intramolecular and intermolecular integrated recombination by wild-type Int by purified IHF protein. (A) Schematic representation of substrate vectors incubated with or without IHF prior to transfection into HeLa cells transiently expressing wild-type Int or Int-h. (B) At 48h after transfection, the proportion of GFP-expressing cells was analyzed by FACS. The ratio of these ratios is plotted against the activation of recombination by IHF. The figure shows the mean of three analyses and the standard deviation indicated by the vertical line. The actual mean (%) of GFP-expressing cells in the presence and absence of IHF were, respectively, intramolecular recombinant Int (7.93/1.26), Int-h (17.57/13.14); intermolecular recombination Int (13.94/3.47), Int-h (20.33/16.83).

FIG. 5 shows an example of an expression vector designed for sequence-specific DNA recombination in CHO-DG44 cells. "P/E" refers to a combined unit comprising enhancer and promoter elements, "P" is a promoter element, and "T" is a transcription termination site necessary for polyadenylation of transcribed mRNA. "GOI" refers to the target gene, "dhfr" refers to the amplifiable selectable marker dihydrofolate reductase, "FP" refers to the fluorescent protein, e.g., ZsGreen, "npt" refers to the selectable marker neomycin phosphotransferase. The arrow indicates the transcription start point in the transcription unit. Sequence-specific recombination between the recombination sites attP or attB located on the first DNA and attP or attB located on the second DNA is indicated by the cross-hatching and is mediated by the bacteriophage lambda integrase. "att" refers to the attachment sites exemplarily shown formed by recombination between attP and attP, attP and attB, attB and attP, or attB and attB, respectively, of the first and second DNAs.

The terms "transformation" or "to transform", "transfection" or "to transfect" as used herein refer to any introduction of a nucleic acid sequence into a cell that results in a genetically modified, recombinant, transformed or transgenic cell. Introduction is performed by any method known in the art and as described in Sambrook, j.et al (1989) Molecular Cloning: a Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Ausubel, F.M.et al. (1994 updated) Current protocols in Molecular Biology, New York: the method described by Greene Publishing Associates and Wiley-Interscience. Such methods include, but are not limited to, lipofection, electroporation, polycation (e.g., DEAE-dextran) -mediated transfection, protoplast fusion, viral infection, and microinjection, or by calcium method, electroshock method, intravenous/intramuscular injection, spray method (aerolysis), or oocyte injection. Transformation can result in transient or stable transformation of the host cell. The term "transformation" or "to transform" also refers to the introduction of a viral nucleic acid sequence in the respective native manner of the respective virus. The viral nucleic acid sequence need not be present as a naked nucleic acid sequence, but may be packaged within a viral protein envelope. Thus, the term does not relate only to the methods generally known under the term "transformation" or "carrying out transformation". Transfection methods which provide optimal transfection frequency and expression of the introduced nucleic acid are preferred. Suitable methods may be determined using routine procedures. For stable transfectants, the construct is either integrated into the host cell genome or the artificial chromosome/minichromosome or is present in episomal form for stable maintenance in the host cell.

The term "recombination sequence" as used herein relates to attB, attP, attL and attR and derivatives thereof. Examples of attB sequences are shown in SEQ ID NO: 13, an example of attP sequence is shown in SEQ id no: specifically shown in fig. 14, an example of attL sequence is shown in SEQ ID NO: 15, examples of attR sequences are shown in SEQ ID NO: 16 are shown in detail.

The term "derivative" as used herein relates to attB, attP, attL and attR sequences having one or more, preferably 7, more preferably 2, 3, 4, 5 or 6 substitutions in the overlap region and/or core region compared to the naturally occurring attB, attP, attL and attR sequences. The term "derivative" also relates to at least one core Int binding site plus one or more copies of the Int arm binding site of attP, attL or attR. The term "derivative" also relates to at least one core Int binding site plus one or more copies of an IHF, FIS or XIS factor binding site of attP, attL or attR. The term "derivative" also relates to combinations of these features. The term "derivative" furthermore relates to any functional fragment thereof as well as to endogenous nucleotide sequences in eukaryotic cells that support sequence-specific recombination, such as attH identified in the human genome (see, for example, WO 01/16345). The term "derivative" generally includes attB, attP, attL or attR sequences suitable for the intended use of the invention, which means that these sequences mediate sequence-specific recombination events driven by bacteriophage lambda integrase (wild-type or modified).

The term "functional fragment" relates to attB, attP, attL and attR sequences (including the presence or absence of wild-type or modified protein binding sites) having substitutions, deletions and/or insertions which do not significantly affect the use of said sequences in recombination events driven by wild-type or modified bacteriophage lambda integrase. Compared to the corresponding naturally occurring recombination sequence, with the same recombinase under the same conditions (e.g., in vitro or in vivo use, same host cell type, same transfection conditions, presence or absence of the same host factor, same buffer conditions, same temperature, etc.), functionality is not significantly affected when the recombination frequency is at least about 70%, preferably at least about 80%, more preferably about 90%, even more preferably at least about 95%, and most preferably more than 100%. Or substitution, deletion and/or insertion of attB, attP, attL and/or attR sequences confers at least one promoting effect on the recombination event driven by the wild-type or modified lambda phage integrase, which promoting may include, for example, (i) increasing the efficiency of the recombination event (integration and/or excision), (ii) increasing the specificity of the recombination, (iii) facilitating excision recombination events, (iv) facilitating integrative recombination events, (v) eliminating the need for some or all of the host factors, compared to the performance of the corresponding native recombination sequences with the same recombinase under the same conditions (see above).

The function of the modified recombination site or modified integrase can be demonstrated by methods that depend on the particular characteristics desired and are known in the art. For example, the cotransfection assay described in the present invention (see conclusion 5.1 or example 3 of WO01/16345) can be used to characterize integrase-mediated extrachromosomal DNA recombination in various cell lines. Briefly, cells are co-transfected with an expression vector encoding an integrase protein and a substrate vector, which is a substrate for the recombinase, encoding a functional/non-functional reporter gene (e.g., a fluorescent protein such as GFP) and containing at least one recombination sequence therein. Once the expression vector expresses integrase, the function of the reporter gene will become non-functional/functional. Thus, the recombination activity can be analyzed by harvesting the recombinant substrate vector and looking for recombination evidence at the DNA level (e.g., by PCR, recombination region sequence analysis, restriction enzyme analysis, Southern blot analysis), or by looking for recombination evidence at the protein level (e.g., ELASA, Western blot analysis, radioimmunoassay, immunoprecipitation, immunostaining, FACS analysis of fluorescent proteins).

The term "overlap region" as used herein defines a sequence in which DNA strand exchanges occur in the recombination sequence, including strand breaks and religation, involving the consensus DNA sequence 5 '-TTTATAC-3' of the wild type att site, or which has functional nucleotide substitutions. The only prerequisite is that the sequences of the overlapping regions between the recombination partner sequences are identical.

The term "core binding site" relates to 2 incompletely repeated copies in each set of wild-type att sites, in inverted orientation, separated by overlapping regions. The core binding site is necessary for recombination to occur with low affinity binding of the recombinase. Each core binding site comprises 9 immediately adjacent base pairs and relates to a DNA sequence which contains a nucleotide sequence of 5 '-CTGCTTTTT-3' in the case of B-sequences at the wild-type att site, a nucleotide sequence of 5 '-CAAGTTAGT-3' in the case of B '-sequences (reverse complement), a nucleotide sequence of 5' -CAGCTTTTT-3 'in the case of C-sequences at the wild-type att site, a nucleotide sequence of 5' -CAACTTAGT-3 'in the case of C' -sequences at the wild-type att site (reverse complement), or which has functional nucleotide substitutions.

The term "Int arm binding site" or "arm binding site" as used herein relates to the consensus sequence 5 '-C/AAGTCACTAT-3', or said sequence having functional nucleotide substitutions. The Int arm binding site can be located at various distances upstream and/or downstream of the core Int binding site.

The terms "homologue" or "homologous" or "similar" as used herein with respect to a recombination sequence, arm-binding site and host factor binding site relate to a nucleic acid sequence having about 70%, preferably about 80%, more preferably about 85%, still more preferably about 90%, further preferably about 95%, most preferably about 99% identity compared to the naturally occurring recombination sequence, arm-binding site and host factor binding site. For example, in the similarity algorithm BLAST at NCBI using standard parameters (Basic Local alignment search Tool, Altschul et al, Journal of Molecular Biology 215, 403-^-5。

The term "vector" as used herein relates to a naturally occurring or synthetically produced construct, such as a plasmid, phagemid, cosmid, artificial chromosome/minichromosome, phage, virus or retrovirus, for uptake, propagation, expression or delivery of a nucleic acid in a cell. Methods for constructing vectors are well known to those skilled in the art and are described in various publications. In particular, techniques for constructing suitable vectors, including descriptions of functional and regulatory components (e.g., promoters, enhancers, terminators and polyadenylation signals, selectable markers, origins of replication and splicing signals), are reviewed in Sambrook, J.et al (1989), supra, and in the references cited therein, in great detail. Eukaryotic expression vectors typically also contain prokaryotic sequences that facilitate propagation of the vector in bacteria, such as an origin of replication and an antibiotic resistance gene for selection in bacteria. Various eukaryotic expression vectors containing cloning sites capable of operably linking polynucleotides are known in the art, and some of these may be obtained from, for example, Stratagene, La Jolla, CA; invitrogen, Carlsbad, CA; commercially available from Promega, Madison, Wis orgD Biosciences Clontech, Palo Alto, Calif.

As used herein, the terms "gene of interest", "desired sequence" or "desired gene" are synonymous and refer to a polynucleotide sequence of any length encoding a product of interest. The selected sequence may be a full-length or truncated gene, a fused or tagged gene, and may be a cDNA, genomic DNA, or a fragment of DNA, preferably a cDNA. It may be the native sequence, i.e. the naturally occurring form, or mutated or modified as desired. These modifications include codon optimization, humanization or labeling which optimizes the codons used in the selected host cell. The selected sequence may encode a secreted, cytoplasmic, nuclear, membrane-bound or cell surface polypeptide. "target product" includes proteins, polypeptides, fragments thereof, peptides, antisense RNA, all of which are expressed in the host cell of choice.

The terms "nucleic acid sequence", "nucleotide sequence" or "DNA sequence" as used herein refer to oligonucleotides, nucleotides or polynucleotides and fragments and portions thereof, as well as DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent either the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence or a mixture of both. Polynucleotides of the invention include a nucleic acid region in which one or more codons are replaced by its synonymous codon.

The nucleic acid sequences of the invention may be prepared using standard techniques well known to those skilled in the art. The term "encode" refers to the inherent property of a particular nucleotide sequence in a nucleic acid (e.g., a gene or mRNA in a chromosome) that serves as a template for the synthesis of other polymers and macromolecules in biological processes having the specified nucleotide sequence (i.e., rRNA, tRNA, other RNA molecules) or amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein when the mRNA it produces is transcribed and translated to produce the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically provided by the sequence listing) and the non-coding strand (which serves as a transcription template) of a gene or cDNA can be used to encode a protein or other product of the gene or cDNA. Nucleic acids encoding a protein include any nucleic acid having a different nucleotide sequence, but which, due to the degeneracy of the genetic code, encodes the same amino acid sequence of the protein. Nucleic acids and nucleotide sequences encoding proteins may include introns.

The term "polypeptide" is used interchangeably with amino acid residue sequence or protein and refers to a polymer of amino acids of any length. These terms also include proteins that are post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation or protein processing. The structure of the polypeptide may be modified or altered, for example by fusion with other proteins, amino acid sequence substitutions, deletions or insertions, while the molecule retains its biological functional activity. For example, specific substitutions in the amino acid sequence of a polypeptide or its nucleic acid coding sequence can result in a protein with similar properties. Amino acid modifications can be made, for example, by site-directed mutagenesis or polymerase chain reaction-mediated mutation of the nucleic acid sequence.

The term "expression" as used herein refers to the transcription and/or translation of a heterologous nucleic acid sequence in a host cell. The level of expression of the desired product in the host cell is determined either by the amount of the corresponding mRNA present in the cell or by the amount of the desired polypeptide encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantified by Northern blot hybridization, RNase RNA protection, in situ hybridization of cellular RNA or by PCR (see Sambrook, J.et al (1989), supra; Ausubel, F.M.et al (1994 updated), supra). The protein encoded by the selected sequence can be quantified by various methods, such as by ELISA, by Western blotting, by radioimmunoassay, by immunoprecipitation, by assay for the biological activity of the protein, or by immunostaining of the protein followed by FACS-analysis PCR (see Sambrook, J.et al (1989), supra; Ausubel, F.M.et al (1994 updated), supra).

An "expression cassette" defines a region of a construct containing one or more genes to be transcribed, wherein the genes comprised by the fragment are operably linked to each other and transcribed from a single promoter, such that different genes are at least transcriptionally linked. One transcription unit can transcribe and express more than one protein and product. Each transcriptional unit includes the regulatory elements necessary for the transcription and translation of any one of the selected sequences contained in the unit.

The term "operably linked" refers to two or more nucleic acid sequences or sequence elements positioned in a manner that allows them to function in the intended manner. For example, a promoter and/or enhancer is operably linked to a coding sequence if it functions in cis to control or regulate the transcription of the linked sequence. Typically, but not necessarily, the operably linked DNA sequences are immediately adjacent and, where necessary to join two protein coding regions or in the case of a secretion leader, must be immediately adjacent and in reading frame.

The term "selectable marker gene" refers to a gene that, when in the presence of a corresponding selection agent, allows only cells carrying the gene to be specifically screened out or eliminated. For example, an antibiotic resistance gene can be used as a positive selection marker gene which allows host cells transformed with the gene to be screened for positive form in the presence of the corresponding antibiotic; under selective culture conditions, non-transformed host cells cannot grow or survive. The selectable marker may be positive, negative or bifunctional. A positive selectable marker allows for selection of cells carrying the marker by conferring drug resistance to the host cell or compensating for metabolic or catabolic deficiencies. Conversely, a negative selectable marker allows cells bearing the marker to be selectively eliminated. For example, using the HSV-tk gene as a marker would sensitize cells to agents such as acyclovir (acyclovir) and ganciclovir (ganciclovir). Selectable marker genes (including amplifiable selectable marker genes) as used herein include recombinantly engineered mutants and variants, fragments, functional equivalents, derivatives, homologs and fusions of the native selectable marker gene, provided that the encoded product retains the selectable properties. Useful derivatives typically have substantial sequence similarity (at the amino acid level) in the region or domain of the selectable marker associated with the selectable trait. A variety of marker genes have been described, including bifunctional (i.e.positive/negative) markers (see, e.g., WO92/08796 and WO 94/28143), incorporated herein by reference. For example, selectable genes commonly used in eukaryotic cells include the Aminoglycoside Phosphotransferase (APH) gene, the hygromycin phosphotransferase (HYG) gene, the dihydrofolate reductase (DHFR) gene, the Thymidine Kinase (TK) gene, the glutamine synthetase gene, the asparagine synthetase gene, and genes encoding neomycin resistance (G418), puromycin, histidinol D, bleomycin, and phleomycin.

Screening can also be performed by Fluorescence Activated Cell Sorting (FACS) using, for example, cell surface markers, bacterial β -galactosidase or fluorescent proteins (e.g., Green Fluorescent Protein (GFP) and its variants from jellyfish (Aequorea victoria) and sea cucumber (Renilla reniformis) or other species; Red fluorescent protein, fluorescent protein and its variants from non-bioluminescent species (e.g., sea anemone pseudoscirpus (Discosoma sp.), sea anemone (Anemonia sp.), Eucalyptus (Clavularia sp.), and Corallium japonicum (Zoanthus sp.)) to screen for recombinant cells.

The term "selective agent" refers to a substance that interferes with the growth or survival of a host cell lacking a particular selectable gene. For example, transfected cells are screened for the presence of an antibiotic resistance gene, such as APH (aminoglycoside phosphotransferase), using an antibiotic geneticin (G418).

Lambda phage integrase (commonly and herein designated "Int"), like Cre and Flp, belongs to the integrase family of sequence-specific conserved DNA recombinases. The natural function of Int is to catalyze the integrative recombination between two different recombination sequences, called attB and attP. AttB contains 21 nucleotides and was initially isolated from the e.coli genome; mizuuchi, M.and Mizuuchi, K. (1980) Proc.Natl.Acad.Sci.USA, 77, pp.3220. On the other hand, attP with 243 nucleotides is much longer and occurs naturally in the lambda phage genome; landy, A.and Ross, W. (1977) Science, 197, pp.1147. The Int recombinase has 7 binding sites in attP and 2 binding sites in attB. The biological function of Int is the sequence-specific integration of the circular phage genome into the attB site on the E.coli chromosome. Int requires a protein cofactor, the so-called integration host factor (commonly and herein designated as "IHF") for integrated recombination; kikuchi, y.und Nash, h. (1978) j.biol.chem., 253, 7149. IHF is required for the assembly of functional recombinant complexes with attP. The second cofactor for this integration reaction is the DNA negative supercoiled of attP. Finally, recombination between attB and attP leads to the formation of two new recombination sequences, called attL and attR, which serve as substrates and recognition sequences for further recombination reactions, i.e.cleavage reactions. For example, Landy, A. (1989) Annu. Rev. biochem., 58, pp.913 provides a comprehensive overview of lambda phage integration.

Excision of the phage genome from the bacterial genome is also catalyzed by the Int recombinase. For this, in addition to Int and IHF, another cofactor encoded by the lambda phage is required. It is an excisionase (commonly and herein designated "XIS") with two binding sites at attR; gottesman, M.and Weisberg, R. (1971) The Bacteriophage Lambda, Cold Spring Harbor Laboratory, pp.113. In contrast to integrative recombination, a DNA negative supercoiling of the recombination sequences is not necessary for excisional recombination. However, DNA negative supercoils increase the efficiency of this recombination reaction. Further improvement of the efficiency of the excision reaction can be achieved by a second cofactor, known as FIS (inverted stimulator), which acts in combination with XIS; landy, a. (1989) annu.rev.biochem., 58, pp.913. The excision reaction is genetically the exact reverse of the integration reaction, i.e., the regeneration of attB and attP. For example, Landy, A. (1989) Annu. Rev. biochem., 58, pp.913 provides a comprehensive overview of lambda phage excision.

One aspect of the invention relates to a method for DNA sequence specific recombination in eukaryotic cells comprising

a) Introducing a first attB, attP, attL or attR sequence or derivative thereof into the cell,

b) introducing a second attB, attP, attL or attR sequence or derivative thereof into the cell, wherein when said first DNA sequence comprises an attB sequence or derivative thereof said second sequence comprises an attB, attL or attR sequence or derivative thereof, or when said first DNA sequence comprises an attP sequence or derivative thereof said second sequence comprises an attP, attL or attR sequence or derivative thereof, or when said first DNA sequence comprises an attL sequence or derivative thereof said second sequence comprises an attB, attP or attL sequence or derivative thereof, or when said first DNA sequence comprises an attR sequence or derivative thereof said second sequence comprises an attB, attP or attR sequence or derivative thereof,

c) sequence-specific recombination was performed by the lambda phage integrase Int.

Preferably, step c) of the method is sequence specific recombination by Int or by Int and XIS, FIS, and/or IHF. More preferably, step c) of the method is sequence-specific recombination by Int or by Int and XIS factor, or by Int and IHF, or by Int and XIS and IHF. It is further preferred that step c) of the method is a sequence specific recombination by a modified Int, preferably Int-h or Int-h/218. Depending on the context, the use of modified Int together with XIS, FIS and/or IHF is also included within the meaning of the present invention.

In a more preferred embodiment of the method, the DNA sequence-specific recombination in the eukaryotic cell is carried out between identical or nearly identical recombination sites. Accordingly, the present invention relates to the above sequence specific recombination process, wherein when said first DNA sequence comprises an attB sequence or a derivative thereof, said second sequence also comprises an attB sequence or a derivative thereof, or when said first DNA sequence comprises an attP sequence or a derivative thereof, said second sequence comprises an attP sequence or a derivative thereof, or when said first DNA sequence comprises an attL sequence or a derivative thereof, said second sequence comprises an attL sequence or a derivative thereof, or when said first DNA sequence comprises an attR sequence or a derivative thereof, said second sequence comprises an attR sequence or a derivative thereof.

The methods of the present invention can be performed with not only naturally occurring attB, attP, attL, and/or attR sequences, but also modified, e.g., substituted attB, attP, attL, and/or attR sequences. For example, integrated recombination between attB and attP homologous sequences (mutants of wild-type sequences) of lambda phage and E.coli has been observed, wherein attB has one or more substitutions (Nash, H. (1981) Annu.Rev.Genet., 15, pp.143; Nussinov, R.and Weisberg, R. (1986) J.biomol.Structure.dynamics, 3, pp 1134) and/or in attP (Nash, H. (1981) Annu.Rev.Genet., 15, pp.143).

Accordingly, the present invention relates to a method wherein the attB, attP, attL, and/or attR sequences used are compared with the naturally occurring attB, attP, attL, and/or attR sequences with one or more substitutions. Preferred methods have 1, 2, 3, 4, 5, 6, 7 or more substitutions in attB, attP, attL, and/or attR sequences. The substitution may occur not only in the overlap region but also in the core region. The complete overlap region containing 7 nucleotides may also be substituted. More preferred methods introduce substitutions in either the core region of the attB, attP, attL, and/or attR sequences or in their overlapping regions. Preferably one substitution is introduced in the overlap region and simultaneously one or two substitutions are introduced in the core region. The invention also relates to a method wherein the attB, attP, attL, and/or attR sequences used are derivatives of the recombination site, including functional fragments thereof, compared to the naturally occurring attB, attP, attL, and/or attR sequences.

The recombinant sequence is selected to have one or more modifications in place of this form, so that recombination can occur despite the modifications. Examples of such substitutions are listed, without limitation, in publications such as Nash, h. (1981), supra and Nussinov, r.and Weisberg, r. (1986), supra. Further modifications can be readily introduced by, for example, mutagenesis methods (Ausubel, F.M.et al. (1994 updated), some of which are described above) and their use can be tested by, for example, test recombinations as described in the examples of the invention (examples 1 and 2, conclusion 5.1).

Furthermore, the present invention relates to a method wherein the used attB, attP, attL, and/or attR sequences contain only one respective core Int binding site, however, it is also preferred to contain more than 2 core Int binding sites. In a preferred embodiment, the present invention relates to a method wherein the attB, attP, attL, and/or attR sequences used consist of only one respective core Int binding site. In another preferred embodiment, the attB, attP, attL, and/or attR sequences used consist of two or more core Int binding sites.

The invention further relates to a method wherein the used attB, attP, attL, and/or attR sequence contains, in addition to the core Int binding site, one or more, preferably 2, 3, 4, 5, or more than 5 copies of the Int arm binding site. The binding site comprises a consensus motif having the sequence 5 '-C/AAGTCACTAT-3' (SEQ ID NO: 1), or a modified sequence thereof having nucleotide substitutions as well as Int binding functions. The arm binding sites of Int may be located at various distances upstream and/or downstream of the core Int binding site.

To carry out the method of the invention, the first recombination sequence may contain additional DNA sequences which allow integration at a desired target locus, such as a locus in the genome of a eukaryotic cell or an artificial/minichromosome. Such recombination occurs, for example, via homologous recombination mediated by intracellular recombination mechanisms. For such recombination, the additional DNA sequence must be homologous to the DNA of the target locus and located at the 3 'and 5' ends of attB, attP, attL, or attR sequences or derivatives thereof, respectively. The skilled person knows how much homology must be, how long the respective 3 'and 5' sequences must be in order for homologous recombination to occur with sufficient probability, see reviews by Capecchi, M. (1989) Science, 244, pp.1288.

However, the first recombination sequence may also be integrated into the eukaryotic cell genome or any artificial/minichromosome by other mechanisms, e.g., via random recombination also mediated by intracellular recombination events. Integration of said first recombination site via sequence-specific recombination is also conceivable using a site different from the site of integration, for example with loxP/FRT sequences.

The second recombination sequence may also comprise a DNA sequence necessary for integration into the desired target locus via homologous recombination. For the methods of the invention, the first and/or second recombination sequences may comprise additional DNA sequences. Preference is given to a process in which both DNA sequences comprise a further DNA sequence.

The first and second sequences, with or without additional DNA sequences, may be introduced in a sequential manner or in a co-transformed manner, wherein the recombination sequences are located on two different DNA molecules. Preferably, the method is one wherein the first and second DNA, with or without additional DNA sequences, are located on and introduced into a single DNA molecule in a eukaryotic cell. Furthermore, it is possible to introduce the first recombination sequence into a cell and the second recombination sequence into another cell, followed by cell fusion. The term fusion refers to biological hybridization and to the cell fusion in the broadest sense.

The method of the present invention can be used, for example, to invert a DNA fragment between recombination sequences in opposite directions (indecters) in intramolecular recombination. Furthermore, the method of the present invention can also be used to delete DNA fragments between recombination sequences in the same orientation in intramolecular recombination. If each recombination sequence is integrated in the 5 '-3' or 3 '-5' orientation, they are in the same orientation. If, for example, attB sequences are integrated in the 5 '-3' direction and attP sequences are integrated in the 3 '-5' direction, the recombination sequences are in the opposite direction. If each recombination sequence is integrated into the intron sequences 5 'and 3' to the exons, for example via homologous recombination, and the integration is carried out by integrase, in the case of reverse recombination sequences the exons will be inverted, and in the case of homologous recombination sequences the exons will be deleted. In this process, the polypeptide encoded by the respective gene may lose its activity or function, or such inversion or deletion may terminate transcription, so that no (intact) transcript is produced. In this way, for example, the biological function of the encoded polypeptide can be studied. In addition, inversion or deletion reactions can also be used to activate expression of a gene encoding a desired polypeptide, for example, by functionally linking the open reading frame encoding the polypeptide to regulatory elements that allow transcription and/or translation of the encoded polypeptide. These regulatory elements include, but are not limited to, promoters and/or promoter/enhancer elements, all of which are known in the art to be suitable for use in different eukaryotic expression systems.

However, the first and/or second recombination sequences may comprise further nucleic acid sequences encoding one or more polypeptides/products of interest. For example, structural, enzymatic or regulatory proteins can be introduced via recombination sequences into the genome which are expressed transiently or stably after intramolecular recombination. The introduced polypeptide/product may be endogenous or exogenous. In addition, marker proteins or biopharmaceutical related therapeutic polypeptides may also be introduced. Those skilled in the art will appreciate that this listing of applications of the method of the present invention in this application is by way of example only and not by way of limitation. Examples of applications according to the invention with the Cre and Flp recombinases currently used may be found, for example, in the reviews by Kilby, N.et al, (1993), Trends Genet, 9, pp.413.

In addition, the method of the invention can be used to delete or invert DNA fragments on vectors by intramolecular recombination on episomal substrates. Deletion reactions can be used, for example, to delete packaging sequences from so-called helper viruses. This method is widely used in industrial production of viral vectors for gene therapy; hardy, s.et al., (1997), j.virol., 71, pp.1842.

Intermolecular recombination fuses two DNA molecules, each containing one copy of each of attB, attP, attL, or attR, or att sequences in various combinations, or derivatives thereof. For example, attB or a derivative thereof may first be introduced into a cell via homologous recombination into a known, well-characterized locus or artificial/minichromosome. Subsequently, the vector or DNA fragment with attB, attP, attL, or attR can be integrated into the genomic attB sequence via intermolecular recombination. In this method, it is preferred that a mutant integrase, such as Int-h or Int-h/218, is co-expressed in the eukaryotic cell in which the recombination has taken place. More preferably, the mutated integrase Int-h/218 is co-expressed. The gene encoding any of the mutant integrases described may be located on a second DNA vector which is transfected, preferably co-transfected, or on a vector or DNA fragment carrying attP, attL, attR or also attB sequences or derivatives thereof. Additional sequences may be located on the vector or DNA fragment with attB, attP, attL, or attR, such as the gene for a specific marker protein flanked by loxP/FRT sequences. This can be achieved, for example, by carrying out comparative expression analysis of different genes in a cell type, which genes are not positively or negatively influenced by the respective integration locus. In addition, the method of the present invention can be used to fuse DNA fragments on a vector by intermolecular recombination on an episomal substrate. Fusion reactions can be used, for example, to express recombinant proteins or related domains to screen for phenotypes. This method can be used for high throughput analysis of protein function in eukaryotic cells and is therefore of considerable importance.

As described above, intermolecular recombination can be used to introduce one or more genes of interest encoding one or more desired polypeptides/products into, for example, episomal substrates, artificial/minichromosomes, or various host cell genomes containing the first recombination sequence. Depending on the context, the second DNA contains, in addition to at least one recombination sequence, such as attP, attB, attL, attR or any derivative thereof, one or more expression cassettes for the expression of one or more desired proteins/products. The expression cassette may be introduced into the desired target locus via a recombination sequence such that sequence-specific recombination occurs between the DNA containing the second recombination sequence and the expression cassette and the first recombination sequence previously introduced into the episomal, artificial/minichromosome or host cell genome. This embodiment can be of considerable importance for the construction of high expression cell lines suitable for the production of biopharmaceutical products.

Depending on the context, the first DNA comprising at least one recombination sequence is introduced into the genome of the host cell or into an artificial/minichromosome or episomal substrate comprised in the host cell, e.g.by random integration. Alternatively, the host cell may be transformed with an artificial/minichromosome or episomal substrate containing the corresponding at least one recombination site. Another method for integrating the recombination sequences into the desired target locus recognized by lambda phage integrase Int is to use homologous recombination techniques as described above.

To facilitate selection of stable transformants for introduction of the recombinant sequence into the desired target locus, a selectable marker gene is co-introduced into the same target locus at the same time. This can be achieved, for example, if the recombination sequence and the selectable marker gene are co-located on the same vector or DNA fragment, they can be introduced into the target locus, for example, by any of the methods described above (homologous recombination, random integration, etc.). Since the expression level of the selectable marker gene is correlated with the transcriptional activity of the integration site, cells showing high expression levels of the integration site, cell durability (robustness) and good growth characteristics, for example, in a bioreactor, can be identified very efficiently. The expression level of a selectable marker gene can be determined by methods known in the art, for example, based on the amount of the corresponding mRNA present in the cell, or based on the amount of polypeptide encoded by the gene. For example, mRNA transcribed from an introduced gene sequence can be quantified by Northern blot hybridization, RNase RNA protection, in situ hybridization of cellular RNA or by PCR (see Sambrook et al, 1989; Ausubel et al, 1994, supra). The protein encoded by the selected sequence may also be quantified by various methods, such as by ELASA, by Western blotting, by radioimmunoassay, by immunoprecipitation, by assay for the biological activity of the protein, immunostaining of the protein followed by FACS analysis, or by measuring the fluorescent signal of a fluorescent protein (see Sambrook et al, 1989; Ausubel et al, 1994 update, supra). In this way, excellent candidates for biopharmaceutical production cell lines can be obtained.

The integrated recombination sequence (first recombination sequence) allows integration of another DNA molecule, for example a vector or a DNA fragment with at least one further recombination sequence (second recombination sequence), into the transcriptionally active locus via sequence-specific recombination with bacteriophage lambda integrase Int. Preferably, the further DNA molecule comprising at least one second recombination sequence further comprises an expression cassette for expression of at least one biopharmaceutical-associated target gene. To this end, a host cell containing a first integrated recombination sequence, preferably at a transcriptionally active locus, integrated into the genome of the host cell, is transfected with a DNA molecule containing a second recombination sequence of bacteriophage lambda integrase Int and cultured under conditions such that sequence-specific recombination between the first and the second recombination sequences is possible, preferably such that the DNA molecule containing the second recombination sequence is integrated into the genome of the host cell containing the first recombination sequence. The first and second recombination sequences may be attP, attB, attL, attR or any derivative thereof which allows sequence-specific recombination by the bacteriophage lambda integrase Int or any functional mutant thereof. For example, if the first recombination sequence contains attP or a derivative thereof, the second may contain attP, attB, attL, attR or any derivative thereof.

Preferred are methods wherein sequence-specific recombination is carried out by Int, or by Int and XIS, FIS and/or IHF. More preferred is a method wherein sequence-specific recombination is performed by Int or by Int and XIS factor, or by Int and IHF, or by Int and XIS and IHF. Further preferred is a method wherein sequence-specific recombination is performed by a modified Int, preferably Int-h or Int-h/218. Depending on the context, the use of modified Int together with XIS and/or IHF is also included within the meaning of the present invention.

In this way, any DNA sequence containing the second recombination sequence of bacteriophage lambda integrase Int can be integrated into a well-defined and defined locus known to the host cell. For the selection of cells in which sequence-specific recombination has taken place, non-functional expression cassettes can be introduced, for example containing a selectable marker gene, for example expression cassettes without promoter or promoter/enhancer or with only partial coding region of the gene. Only when sequence-specific recombination has occurred will a complete and functional expression cassette be generated which efficiently expresses the selectable marker gene, and cells with the target gene integrated via sequence-specific integration can therefore be screened.

By the method of the invention, a production cell line can be obtained which differs from the host cell only in the identity (identity) of the DNA sequence integrated at the designated integration site, e.g.a genomic locus. Because of the less genetic variation between different cell clones, more genetic processes can be used to develop production cell lines, which can reduce the time and effort for clone screening and research to optimize the production process. Such producer cell lines can be used to produce the desired polypeptide.

Thus, another aspect of the invention relates to a method for expressing at least one gene of interest encoding one or more desired polypeptides/products in a eukaryotic cell, comprising:

a) introducing a first DNA comprising an attB, attP, attL or attR sequence or derivative thereof into a cell;

b) introducing a second DNA comprising attB, attP, attL or attR sequences or derivatives thereof and at least one target gene into the cell,

c) contacting said cell with a lambda phage integrase Int;

d) sequence-specific recombination by bacteriophage lambda integrase Int, wherein the second DNA is integrated into the first DNA; and

e) culturing the cell under conditions that allow expression of the gene of interest.

In a preferred method, when said first DNA sequence comprises an attB sequence or a derivative thereof, said second sequence comprises an attB, attL or attR sequence or a derivative thereof, or when said first DNA sequence comprises an attP sequence or a derivative thereof, said second sequence comprises an attP, attL or attR sequence or a derivative thereof, or when said first DNA sequence comprises an attL sequence or a derivative thereof, said second sequence comprises an attB, attP or attL sequence or a derivative thereof, or when said first DNA sequence comprises an attR sequence or a derivative thereof, said second sequence comprises an attB, attP or attR sequence or a derivative thereof.

In a more preferred embodiment of this method, the first DNA has been integrated into the genome, artificial chromosome/minichromosome or episomal element of the host cell, preferably at a site exhibiting high transcriptional activity, prior to introduction of said second DNA into said cell.

The present invention also relates to a method for expressing at least one or more genes of interest in a host cell, wherein said host cell comprises one attB, attP, attL or attR sequence or a derivative thereof integrated into the genome of said host cell, said method comprising:

a) introducing into said cells a DNA containing attB, attP, attL or attR sequences or derivatives thereof and at least one target gene,

b) contacting said cell with a lambda phage integrase Int;

c) sequence-specific recombination by bacteriophage lambda integrase Int, wherein the second DNA is integrated into the first DNA;

d) culturing the cell under conditions that allow expression of the gene of interest.

This method can be carried out not only with the attB, attP, attL or attR sequences or derivatives thereof integrated into the genome of the host cell by gene-redirection of said cell, but also with naturally occurring recombination sequences in the genome, such as the attH site described in WO01/16345 (5'-GAAATTCTTTTTGATACTAACTTGTGT-3'; SEQ ID NO: 17) or any other recombination sequence allowing sequence-specific recombination mediated by Int or functional mutants thereof.

In a preferred method, said sequence-specific recombination is performed by Int or by Int and XIS factor, or by Int and IHF, or by Int and XIS and IHF. More preferred methods are those wherein said sequence-specific recombination is carried out by a modified Int, preferably Int-h or Int-h/218. Depending on the context, the use of modified Int together with XIS, and/or IHF is also included within the meaning of the present invention. Int, Int-h or Int-h/218, XIS and/or IHF can be added to the cells in purified form or co-expressed by the host cells for sequence-specific recombination.

Another embodiment of the above method relates to a method wherein the protein/product encoded by the gene of interest expressed in the host cell is isolated from the cell or, if it is secreted into the culture medium, from the cell culture supernatant.

The production cells are preferably cultured in serum-free medium and in suspension under conditions conducive to expression of the desired gene and to isolation of the protein of interest from the cells and/or cell culture supernatant. Preferably, the protein of interest is recovered from the culture medium as a secreted polypeptide or, if the protein of interest is expressed without a secretion signal, from a host cell lysate. It is necessary to purify the protein of interest from other recombinant proteins, host cell proteins and contaminants in order to obtain a substantially homogeneous preparation of the protein of interest. The first step is usually the separation of the cells and/or particulate cell debris from the culture medium or lysate. The target product is thereafter purified from contaminating soluble proteins, polypeptides and nucleic acids by fractionation, e.g.by immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography on silica or cation exchange resins such as DEAE. Generally, methods are known in the art that teach a skilled artisan how to purify heterologous proteins expressed by a host cell. For example Harris et al (1995) Proteinpurification: a Practical Approach, Pickwood and Hames, eds., IRL Press and scopes, R. (1988) Protein Purification, Springer Verlag describes such a method. Thus, the above-described method of expressing at least one gene of interest may be supplemented with an additional purification step wherein the desired polypeptide is purified from the host cell or, if secreted into the culture medium, from the cell culture.

The method of the invention can be carried out in all eukaryotic cells. The cells and cell lines can be present, for example, in cell culture, and include, but are not limited to, eukaryotic cells, such as yeast, plant, insect, or mammalian cells. For example, the cell may be an oocyte, an embryonic stem cell, a hematopoietic stem cell or any type of differentiated cell. Preferred are methods wherein the eukaryotic cell is a mammalian cell. More preferred is a method wherein the mammalian cell is a human, simian, murine, rat, rabbit, hamster, goat, bovine, ovine or porcine cell. Preferred biopharmaceutical-producing cell lines or "host cells" are human, murine, rat, monkey or rodent cell lines. More preferably hamster cells, preferably BHK21, BHK TK^-CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1 and CHO-DG44 cells or derivatives/progeny of any of said cell lines. CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 are particularly preferred, and CHO-DG44 and CHO-DUKX cells are even more preferred. Furthermore, murine myeloma cells, preferably NS0 and Sp2/0 cells or derivatives/progeny of either of these cell lines are also known as producer cell lines.

Such host cells are most preferred when constructed, adapted and cultured completely under serum-free conditions and optionally in a medium that does not contain any animal-derived proteins/polypeptides. Commercial media, such as Ham ' S F12(Sigma, Deisenhofen, Germany), RPMI-1640(Sigma), Dulbecco ' S modified Eagle ' S medium (DMEM; Sigma), minimal essential medium (MEM; Sigma), Iscove ' S modified Dulbecco ' S medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-SFMII (Invitrogen), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are examples of suitable nutrient solutions. Any of the media may be supplemented, if necessary, with various compounds, examples of which are hormones and/or other growth factors (e.g. insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g. sodium chloride, calcium, magnesium, phosphate), buffers (e.g. HEPES), nucleosides (e.g. adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics, trace elements. Any other necessary additives may also be included in appropriate concentrations well known to those skilled in the art. In the present invention, serum-free medium is preferably used, but a medium supplemented with an appropriate amount of serum may be used to culture the host cells. To culture and select for genetically modified cells expressing a selectable gene, the medium is supplemented with a suitable selective agent.

"desired protein/polypeptide" or "target protein/polypeptide" in the present invention is exemplified by, but not limited to, insulin-like growth factor, hGH, tPA, cytokines, such as Interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, Interferon (IFN) - α, IFN β, IFN γ, IFN ω, IFN σ, Tumor Necrosis Factor (TNF), such as TNF α, TNF β, TNF γ, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF. Also included are preparations of erythropoietin or any other hormone growth factor and any other polypeptide which can act as an agonist or antagonist and/or which has therapeutic or diagnostic utility. The method of the invention can also be advantageously used for the production of antibodies, such as monoclonal, polyclonal, multispecific and single-chain nature antibodies or fragments thereof, e.g., Fab ', F (ab ') 2, Fc and Fc ' -fragments, immunoglobulin heavy and light chains and their constant, variable or hypervariable regions, and Fv-and Fd-fragments (Chamov, S.M.et al (1999) Antibody Fusion Proteins, Wiley-Liss Inc.).

Fab fragments (antigen binding fragments ═ Fab) consist of the variable regions of the two chains, which are held together by adjacent constant regions. They can be formed by digestion of conventional antibodies with, for example, papain, but similar Fab fragments can also be prepared by genetic engineering. Additional antibody fragments include F (ab') 2 fragments that can be prepared by proteolysis with pepsin.

Using genetic engineering methods, it is possible to produce shortened antibody fragments consisting only of the heavy (VH) and light (VL) chain variable regions. They are called Fv fragments (variable fragments-fragments of the variable part). Because these Fv fragments lack covalent linkage of the two chains by the cysteine of the constant chain, Fv fragments are often stabilized. It is advantageous to use short peptide fragments, for example, of 10 to 30 amino acids, preferably 15 amino acids, to link the variable regions of the heavy and light chains. In this way a single peptide chain can be obtained in which VH and VL are linked by a peptide linker. This antibody protein is known as single chain fv (scFv). Examples of such scFv-antibody proteins known in the prior art are described by Huston c.et al (1988) proc.natl.acad.sci.usa, 16, pp.5879.

In recent years, various strategies have been developed to prepare scFv in multimeric derivative form. This is particularly for the preparation of recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with improved binding affinity. To achieve scFv multimerization, scFv were prepared as fusion proteins with multimerization domains. Such a multimerization domain may be, for example, the CH3 region of IgG or a coiled-coil structure (helix structure), such as the leucine zipper region. However, there are also strategies in which multimerization is achieved using the interaction between the VH/VL regions of the scFv (e.g., diabodies, triabodies and pentabodies antibodies). Diabodies (diabodies) are understood by the skilled person to mean derivatives of scFv in a bivalent, homodimeric form. Shortening the linker in the scFv molecule to 5-10 amino acids results in the formation of homodimers in which interchain VH/VL overlap (hypermethylation) occurs. Diabodies can also be constructed by incorporating disulfide bridges. Examples of diabody-antibody proteins of the prior art can be found in Perisic, O.et al (1994) Structure, 2, pp.1217.

Microbodies (minibodies) refer to scFv derivatives in a bivalent, homodimeric form to the skilled person. It consists of a fusion protein comprising as dimerization region the CH3 region of an immunoglobulin, preferably IgG, more preferably IgG1, which is linked to the scFv via a hinge region (e.g. also from IgG1) and a linker region. Examples of microbody-antibody proteins of the prior art can be found in Hu, s.et al (1996) Cancer res, 56, pp.3055.

Trisomy (triabody) refers to the trivalent, homotrimeric form of scFv derivatives to the skilled person (Kortt A. et al (1997) Protein Engineering, 10, pp.423). ScFv derivatives in which VH-VL is directly fused without a linker sequence lead to the formation of trisomy.

The person skilled in the art is also familiar with so-called miniantibodies (miniantibodies) which have a bivalent, trivalent or tetravalent structure and are derived from scFv. Multimerization is carried out by means of dimeric, trimeric or tetrameric coiled coils (Pack, p.et al (1993) Biotechnology, 11, pp.1271; Lovejoy, b.et al (1993) Science, 259, pp.1288; Pack, p.et al (1995) j.mol.biol., 246, pp.28). In a preferred embodiment of the invention, the target gene encodes any one of the above desired polypeptides, preferably a monoclonal antibody, derivative or fragment thereof.

To carry out any of the embodiments of the invention, the integrase must act on the recombination sequence. The integrase or integrase gene and/or the cofactor or cofactor gene, for example factor XIS or factor XIS gene and/or IHF gene, may already be present in the eukaryotic cell before the introduction of the first and second recombination sequences. They may also be introduced between or after the introduction of the first and second recombination sequences. Purification of recombinant enzymes and host factor proteins has been described in the prior art (Nash, H.A. (1983) Methods of Enzymology, 100, pp.210; Filutowicz, M.et al. (1994) Gene, 147, pp.149). If these are unknown, cell extracts or partially purified enzymes, for example by the procedures described in the literature for purification of Int or Cre recombinase, can be used. The purified protein can be introduced into the cells by standard techniques, for example by injection or microinjection or by lipofection as described for IHF in example 2 of the invention. The integrase used for the sequence-specific recombination is preferably expressed in the cells in which the reaction is carried out. For this purpose, a third DNA sequence containing an integrase gene is introduced into the cell. If sequence-specific recombination is carried out, for example, by attL/attR, the XIS factor gene (fourth DNA sequence) can additionally be introduced into the cells. More preferred are methods wherein the third and/or fourth DNA sequences are integrated into the eukaryotic genome of the cell or artificial/minichromosomes via homologous recombination or random means. Such a method is further preferred, wherein the third and/or fourth DNA sequence contains regulatory sequences which lead to spatial expression and/or transient expression of the integrase gene and/or the XIS factor gene.

In this context, spatial expression means that Int recombinase, XIS factor, and/or IHF factor, respectively, are expressed only in specific cell types using cell type-specific promoters, and that recombination is catalyzed only in these cells, such as hepatocytes, kidney cells, nerve cells, or cells of the immune system. When regulating integrase/factor XIS/IHF expression, transient expression can be achieved by activating the promoter at a particular developmental stage or at a particular point in time in an adult organism. In addition, transient expression can also be achieved by using inducible promoters, for example by interferon or tetracycline-dependent promoters; see muller, u. (1999) reviewed in mech. devilop., 82, pp.3.

The integrase used in the process of the invention may be both wild-type and modified (mutated) lambda phage integrase. Since the wild-type integrase is only capable of efficient recombination reactions with cofactors known as IHF, it is preferred to use a modified integrase in the process of the invention. If the method of the invention uses a wild-type integrase, an additional addition of IHF is required to achieve stimulation of the recombination reaction. Modified integrase into which the integrase can be recombined without IHF or other host factors, such as XIS and FIS. For example, recombination reactions between attL and attR sequences can be carried out by modified Int without additional host factors (see conclusion 5.1 and fig. 2C and 2D).

The production of modified polypeptides and screening for desired activity is state of the art and can be readily performed; erlich, h. (1989) PCR technology stockton Press. For example, a nucleic acid sequence encoding a modified integrase is intended to include any nucleic acid sequence that can be transcribed and translated into an integrase in vitro or upon introduction of the coding sequence into a bacterial or eukaryotic cell. The modified integrase protein-encoding sequence may be naturally-occurring (bySpontaneous mutations) or recombinantly engineered mutants and variants, truncated versions and fragments, functional equivalents, derivatives, homologs, and fusions of naturally occurring or wild-type proteins, so long as the encoded polypeptide maintains biological functional activity, i.e., recombinant activity. Recombinant activity is maintained when the modified recombinase enzyme has at least 50%, preferably at least 70%, more preferably at least 90%, most preferably at least 100% of the activity of the wild-type integrase Int as measured in a co-transfection assay with the substrate vector and the expression vector as described in conclusion 5.1 of the present invention or in example 3 of WO 01/16345. Proteins with similar properties can be obtained by substituting the integrase or its nucleic acid coding sequence for some amino acid sequence. Amino acid substitutions providing functionally equivalent integrase polypeptides using the amino acid hydropathy index (Kyte, j.et al (1982) j.mol.biol., 157, pp.105) may be made by performing site-directed mutagenesis or polymerase chain reaction-mediated mutation on the nucleic acid sequence thereof. Preferred mutants or modified integrases according to the invention exhibit improved recombination activity/recombination efficiency or recombination activity which is independent of one or more host factors compared with the wild-type protein. "wild-type protein" refers to a polypeptide encoded by an intact, non-truncated, non-modified, naturally occurring gene. Preferably two lambda phage integrase Int mutants designated Int-h and Int-h/218; miller et al (1980) Cell, 20, pp.721; christ, N.andP. (1999) j.mol.biol., 288, pp.825. In comparison with wild-type Int, Int-h replaces the glutamic acid residue with a lysine residue at position 174. Int-h/218 has another lysine residue at position 218 instead of a glutamic acid residue, and this enzyme is produced by PCR mutation of the Int-h gene. The mutants can catalyze the recombination between attB/attB, attP/attP, attL/attL or attR/attR and all other possible combinations, for example attP/attR, attL/attP, attL/attB or attR/attB or derivatives thereof, in E.coli, in eukaryotic cells and in vitro, i.e.using purified substances in reaction tubes, without cofactors IHF, XIS and/or FIS and negative supercoils. With cofactors, e.g. FIS, mayTo achieve an improvement in recombination efficiency. The mutant Int-h/218 is preferred because this mutant catalyzes the recombination reaction with increased efficiency.

If the first reaction results in excision and the two recombination sequences used are identical, e.g., attP/P, the recombination sequences obtained after recombination will be identical to the sequences on the substrate, e.g., here the two attP sequences. However, if the two partner sequences are different, e.g., attP/R, then the recombination reaction will produce a hybrid recombination sequence that contains a functional half from one sequence (e.g., attP) and half from the other sequence (attR). A functional half-recombination site can be defined as either the 5 'sequence or the 3' sequence of the overlap region, in each case the overlap region is considered to be part of the functional half-site. If the respective overlapping regions of the recombination sequences used are identical, the cleavage reaction can be carried out by any recombination sequence according to the invention. In addition, the overlap region specifies the orientation of the recombination sequences with respect to each other, i.e.inverted or in the same orientation (direct). The reaction proceeds only with low efficiency with wild-type Int, however, the presence of arm binding sites in addition to the core binding site stimulates and increases efficiency with the addition of IHF or without IHF. The reaction can be carried out without any cofactor by the modified Int.

In addition, a method in which another DNA sequence comprising the Xis factor gene is introduced into cells is preferred. More preferably wherein said another DNA sequence further comprises a regulatory DNA sequence which increases the spatial and/or transient expression of the Xis factor gene.

For example, following successful intramolecular integrative recombination (inversion) with Int, which results in activation/inactivation of a gene in a particular cell type, the gene can be inactivated or activated again at a later point in time by inducing spatial and/or transient expression of XIS and simultaneous expression of Int.

In addition, the invention relates to any recombinant sequence or derivative thereof, such as SEQ ID NO: 2, specifically shown in the specification, and an application of the derivative of attP in eukaryotic cell DNA sequence specific recombination. Eukaryotic cells may be present in cell populations of organisms (e.g., mammals) that do not contain integrase or the Xis factor in their cells. The organism can be used to propagate with other organisms that carry integrase or the Xis factor in their cells in order to produce progeny, in whose cells sequence-specific recombination takes place. Therefore, the invention also relates to the application of the integrase or integrase gene and the Xis factor or Xis factor gene and the IHF factor or IHF factor gene in the eukaryotic cell sequence-specific recombination. In addition, the invention relates to eukaryotic cells and cell lines for carrying out the method of the invention, wherein the cells or cell lines are obtained after carrying out the method of the invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, immunology and similar techniques which are within the skill of the art. These techniques are well described in the literature, see, e.g., Sambrook et al, molecular cloning: a Laboratory Manual, 2^nd Ed.，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1989)；Ausubel et al.，Current Protocols in MolecularBiology(1987，updated)；Brown ed.，Essential Molecular Biology，IRL Press(1991)；Goeddel ed.，Gene Expression Technology，Academic Press(1991)；Bothwell et al.eds.，Methods for Cloning and Analysis of Eukaryotic Genes，Bartlett Publ.(1990)；Wu et al.，eds.，Recombinant DNA Methodology，Academic Press(1989)；Kriegler，Gene Transfer and Expression，Stockton Press(1990)；McPherson et al.，PCR：A Practical Approach，IRL Press at OxfordUniversity Press(1991)；Gait ed.，Oligonucleotide Synthesis(1984)；Miller &Calos eds.，Gene Transfer Vectors for Mammalian Cells(1987)；Butler ed.，Mammalian Cell Biotechnology(1991)；Pollard et al.，eds.，Animal Cell Culture，Humana Press(1990)；Freshney et al.，eds.，Culture of Animal Cells，Alan R.Liss(1987)；Studzinski，ed.，Cell Growth and Apoptosis，A Practical Approach，IRL Press at Oxford University Presss(1995)；Melamed et al.，eds.，FlowCytometry and Sorting，Wiley-Liss(1990)；Current Protocols in Cytometry，JohnWiley & Sons，Inc.(updated)；Wirth & Hauser，Genetic Engineering of AnimalsCells，in：Biotechnology Vol.2，Pühler ed.，VCH，Weinheim 663-744；the seriesMethods of Enzymology(Academic Press，Inc.)，and Harlow et al.，eds.，Antibodies：A Laboratory Manual(1987)。

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications cited herein are hereby incorporated by reference in their entirety for the purpose of describing the state of the art to which this invention pertains more fully. The invention as broadly described above will be understood by reference to the following examples, which are included merely for the purpose of illustrating certain examples of the invention and are not intended to limit the invention in any way.

Examples

Method of producing a composite material

1.Preparation of expression vectors and substrate vectors

The construction of mimetics (mock) and Int expression vectors pCMV, pCMVSSInt, pCMVSSInt-h, and pCMVSSInt-h/218 has been documented; lorbach, E.et al (2000) J.mol.biol, 296, pp.1175. Expression of Int is driven by the human cytomegalovirus promoter.

In the moleculeThe substrate vectors used in the recombination experiments, which contain attB/attP (p. lamda. IR) or attL/attR (p. lamda. ER) as direct repeats, are(Promega). The p.lamda.IR was constructed by inserting attB as a double-stranded oligonucleotide into ClaI/EcoRI-cut pPGKneo. The vector is a derivative of pPGKSSInt-h in which the Int is replaced with the neomycin gene (neo) using PstI/XbaI-h gene. The CMV promoter + hybrid intron was generated by PCR using pCMVSSInt as a template and cloned into attB-containing pPGKneo cut with KpnI/ClaI. This CMV-attB-neo-expression cassette was cloned into BamHI-cut by PCRIn (1). The following primers were used

(attP01)5’-GTCACTATCAGTCAAAATACAATCA-3’，(SEQ ID NO：3)

(attP02)5’-TGATTGTATTTTGACTGATAGTGAC-3’，(SEQ ID NO：4)

(PFP-NsiI)5’-CCAATGCATCCTCTGTTACAGGTCACTAATAC-3’，(SEQ ID NO：5)

And (P 'RP-EcoRV-NotI) 5'-ATAAGAATGCGGCCGCAGATATCAGGGAGTGGGACAAAATTGAA-3'(SEQ ID NO: 6) with pGFPattB/attP as template by assembly PCR (assembly PCR) to generate an attP site containing an A → C substitution in its P' -arm that results in the deletion of the translation termination signal (Lorbach, E.et al (2000), supra). The PCR fragment was cut with NsiI and NotI and ligated to the 3' -end of a BamHI/PstI fragment containing a transcription termination cassette generated from pBS302 (Gibco/BRL). The GFP gene as well as the polyA signal were cloned by PCR using pCMVSSGFP (a derivative of pCMVSSInt-h, in which the Int-h gene was replaced with eGFP using PstI/XbaI). The GFP containing PCR fragment was cut with NotI and XbaI and ligated with a BamHI/NotI cut transcription terminator/attP fragment into a BamHI/XbaI cut vector already containing the CMV promoter, attB and the neomycin expression cassette. Construction of p λ ER As with p λ IR, except that pGFPattL/attR (Lorbach, E.et al (2000), supra) was used as a template, attL was generated by PCR and cloned into ClaI/EcoRI cut pPGKneo. Using pGFPattL/attR as template, attR sites were generated by PCR and the products were cleaved with NsiI and NotI.

Between moleculesThe substrate vector used for the recombination assay contained the CMV promoter located before the different attachment sites: pCMVattPmut, the P-arm of which contains 3G → C substitutions. These changes are necessary to delete the ATG initiation codon to prevent GFP expression after recombination. These substitutions are located outside the protein binding site of attP, and they are introduced by assembly PCR. First, two overlapping PCR products were generated, one using the primer pair attP-ATC-1/attP-2 and one using the primer pair attP-ATC-3/attP-4. pGFPattB/attP was used as template. The PCR product was gel-purified as a template for PCR with attP-PstI and attP-XbaI as primers. The product obtained was digested with PstI and XbaI and cloned into pCMVSSInt. The primer sequences for assembling the PCR are

(attP-ATC-1)

5’-TTTGGATAAAAAACAGACTAGATAATACTGTAAAACACAAGATATGCAGTCAC

TA-3’，(SEQ ID NO：7)

(attP-2)5’-TAACGCTTACAATTTACGCGT-3’，(SEQ ID NO：8)

(attP-ATC-3)

5’-CTGCATATCTTGTGTTTTACAGTATTATCTAGTCTGTTTTTTATCCAAAATCTAA-

3’，(SEQ ID NO：9)

(attP-4)5’CTGGACGTAGCCTTCGGGCATGGC-3’，(SEQ ID NO：10)

(attP-PstI)5’-GACTGCTGCAGCTCTGTTACAGGTCAC-3’，(SEQ ID NO：11)

(attP-XbaI)5’-GACTGTCTAGAGAAATCAAATAATGAT-3’(SEQ ID NO：12)。

attB was inserted as a double-stranded oligonucleotide into PstI/XbaI-cut pCMVattPut, yielding pCMVattB. attL was introduced into PstI/XbaI cut pCMVattPmut, using p. lamda.ER as template, to generate pCMVattL by PCR.

The vector containing the transcription termination signal and att site in front of the promoterless GFP gene was constructed as follows: pWSattBGFP was first generated by deleting part of the hygromycin gene from pTKHyg (Clontech) with AvaI and NdeI. The vector backbone was ligated after filling the cohesive ends with Klenow polymerase. The PCR generated attB-GFP fragment was cloned into the MfeI and HindIII sites, creating a new attB 5' NheI site. Finally, a transcription termination sequence was inserted by restriction with EcoRI and NheI. A BamHI/NotI transcription termination-attR fragment was isolated from p λ ER and inserted into pWSattBGFP cut with the same enzyme to generate pWSattRGFP. Using pGFPattP/attB as a template, PCR was performed on the attP site, which was inserted into EcoRI/NotI-cut pWSattBGFP in place of attB, yielding pWSattPGFP. Plasmids were isolated from E.coli XL1-Blue strain by affinity chromatography (Qiagen). After DNA sequencing, the nucleotide composition of the relevant genetic elements was confirmed using the fluorescent 373A system (Applied Biosystems).

2.Cell culture, recombination assays, and flow cytometry

HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 0,1mg/ml streptomycin and 100U/ml penicillin.

A typical recombination assay is performed as follows. Cells were harvested, washed with PBS and resuspended in RPMI 1640 without L-glutamine and phenol red (Life Technologies). Then, 60. mu.g in total of the expression vector and the substrate vector in a molar ratio of 1: 1 were introduced into about 1X 10 at 300V and 960. mu.F using a gene pulser (Genespulse, Bio-Rad)⁷In the cell of (a). After electroporation, cells were plated in 10cm dishes at the appropriate dilution. Single cell suspensions were prepared 24h, 48h and 72h after transfection. Dead cells were removed by staining analysis with 7-amino-actinomycin D (Sigma) and cells were analyzed with FACScalibur (Becton Dickinson). Using CellQuest^TMThe software analyzes the FACS data. In each experiment, the measurement molecule was co-transfected with 40. mu.g pCMV and 20. mu.g pEGFP-C1(Clontech)WorkshopTransfection efficiency of recombination assays; measurement molecules were co-transfected with 30. mu.g pCMV and 30. mu.g pEGFP-C1Inner partTransfection efficiency of recombination assays.

Assays involving purified IHF were performed, as described above, by first introducing 30. mu.g of Int expression vector via electroporation to approximately 6X 10⁶In the cell. After 3 to 4 hours, about 1X 10 transfection with 2. mu.g of substrate vector⁵The cells of (2) are subjected to intramolecular recombination or transfected with a total of 2. mu.g of substrate vector in a molar ratio of 1: 1 by about 1X 10⁵The cells of (a) undergo intermolecular recombination. Substrate and 2. mu.g of purified IHF (Lange-GustafsonBJ, Nash HA., Purification and properties of Int-h, a variant protein involved in dissolved contained in specific binding of bacterial lambda., J Biol chem.1984 Oct 25; 259 (20): 12724-32) were pre-incubated for at least 30min at room temperature in a low salt buffer (50mM NaCl, 10mM Tris-HCl, pH8.0, 1mM EDTA). Transfection of the IHF-DNA complex was achieved with FuGene (Boehringer Mannheim) with an overall efficiency in the range of 80%. After an additional 48h as described above, the cells were analyzed by flow cytometry.

CHO-DG44/dhfr grown in permanent suspension in serum-free CHO-S-SFMII medium (Invitrogen, Carlsbad, Calif.) supplemented with hypoxanthine and thymidine (Invitrogen, Carlsbad, Calif.)^-/-Cells (Urlaub, G.et al., (1983), Cell, 33, pp.405) containing 5% CO at 37 ℃₂In a humidified atmosphere using a cell culture flask. Every 2 to 3 days, cells were cultured at 1-3X 10⁵The cells/mL concentration was inoculated into fresh medium.

CHO-DG44 cells were stably transfected with Lipofectamine Plus reagent (Invitrogen, Carlsbad, Calif.). For each transfection, 0,8mL of 6X 10 in CHO-S-SFMII medium supplemented with hypoxanthine/thymidine (HT)⁵One exponentially growing cell was seeded into a well of a 6-well plate. A total of 1. mu.g plasmid DNA, 4. mu.L Lipofectamine and 6. mu.L Plus reagent in a volume of 200. mu.L were used for each transfection and added to the cells, followed by the manufacturer's experimental procedure. After 3h of culture, 2mL of HT-supplemented CHO-S-SFMII medium was added. In the case of selection based on neomycin phosphotransferase, 2 days after transfection, the medium was replaced with CHO-S-SFMII medium supplemented with HT and 400. mu.g/mL G418(Invitrogen), and the medium was changed every 3 to 4 days for 2 to 3 weeks to select a mixed cell population. For DHFR-based selection of stably transfected CHO-DG44 cells, hypoxanthine/thymidine-free CHO-S-SFMII medium was used. By adding 5-2000nM methotrexate (Sigma, Deisenhofen) to the mediumGermany) as an amplification selection reagent, DHFR-based gene amplification was achieved.

3.sICAM and MCP-1ELISA

Two sICAM-specific monoclonal antibodies were developed in this laboratory (in house), one of which was an HRPO-conjugated antibody (see, e.g., US5,284,931 and 5,475,091), and used to quantitate sICAM titers in supernatants of stably transfected CHO-DG44 cells using ELISA standard procedures (Ausubel, F.M. et al, (1994, updated) Current protocols in molecular biology. New York: Greene Publishing Association and Wiley-Interscience). Purified sICAM protein was used as standard. Samples were analyzed using a Spectra Fluor Plus reader (TECAN, Crailsheim, Germany).

The titer of MCP-1 from the supernatant of stably transfected CHO-DG44 cells was quantified by ELISA using OptEIA human MCP-1 according to the manufacturer's experimental protocol.

Example 1: kinetics of intramolecular and intermolecular recombination reactions

We have shown in previous studies that mutant Int catalyzes intramolecular integration and excision recombination reactions in E.coli and human cells in the absence of native accessory factors (Christ, N.et al (1999), supra; Lorbach, E.et al (2000), supra). However, an interesting problem with respect to the interaction of episomal DNA fragments within mammalian cells is related to the ability of mutant Int to undergo intermolecular recombination, i.e., where the two recombination sites are located in trans on different DNA molecules. Therefore, we first compared intramolecular and intermolecular integrated recombination reactions.

Intramolecular recombination was tested with substrates containing attB and attP as direct repeats flanking the transcription termination signal. The recombination cassette is flanked by a CMV promoter and a GFP coding region. Recombination between attB and attP generates the hybridization sites attL and attR and leads to a cleavage termination signal. Thus, subsequent expression of the GFP gene served as an indicator of recombination (FIG. 2A, top).

HeLa cells were co-transfected with expression vectors for Int, Int-h, or Int-h/218 and substrate vectors. The backbone (mock) of the expression vector was used as a negative control. Transfection efficiencies determined independently for each experiment ranged from 95% to 98% (data not shown). FACS analysis of 3 experiments showed that both mutant Int efficiently catalyze recombination, resulting in about 30% GFP expressing cells in some experiments (fig. 2A, bottom). The nucleotide sequence of the recombinant product was determined indirectly by DNA sequencing of the PCR fragment, and the results demonstrated that the mutant Int-catalyzed strand transfer reaction produced the expected hybrid att sites (data not shown).

It is clear that the double mutant Int-h/218 is more active than Int-h, whereas the wild-type Int is almost inactive. The proportion of GFP expressing cells increased within 48h after transfection and remained stable for the following 24 h. The reaction time course also shows that most recombination events must occur within the first 24 h. This is in good agreement with the time course of expression of Int-h/218 in HeLa cells (data not shown). Although we cannot exclude that the proportion of GFP expressing cells is due to intermolecular, rather than intramolecular, integration of the recombination, the data obtained are still available as reference for our analysis of intermolecular recombination.

Intermolecular integrated recombination was analyzed by placing attB and attP on different plasmids. Recombination translocated the CMV promoter to a position upstream of the GFP gene (fig. 2B, top). Thus, only intermolecular recombination between attB and attP will produce GFP expressing cells. Co-transfection with both substrate vectors with the Int expression vector, followed by FACS analysis, gave results comparable to those produced by intramolecular recombination of the substrates (FIG. 2B, bottom). Likewise, most recombination events must occur within the first 24h after transfection, and Int-h/218 is more active than Int-h. Wild-type Int produces only a very small proportion of GFP expressing cells. These results demonstrate that intramolecular integrative recombination by mutant Int is at least as effective as the corresponding intramolecular recombination reaction over a period of 24 to 72 h.

Next, the intramolecular and intermolecular excision (attL × attR) recombination pathways were compared using the same assay strategy. The results again show that intermolecular recombination by mutant Int is as efficient as intramolecular recombination (FIGS. 2C and D). However, the efficiency of the excisional recombination reaction is slightly reduced compared to the integrative recombination. Also, recombination of wild-type Int was hardly observed.

Example 2: the DNA arm binding site of att is not necessary, but recombination is stimulated

The current results show that mutant Int catalyzes both integrative and excisional recombination of episomal substrates in a significant number of transfected cells. In contrast, the recombination activity of wild-type Int was hardly detectable in the above background. Since the excisional recombination by wild-type Int relies on the presence of the protein cofactors IHF and XIS, but does not require negative DNA supercoils, this result demonstrates that the eukaryotic counterparts of these cofactors are absent in human cells. Furthermore, episomal substrates are known to become topologically relaxed immediately after transfection (Schwikardi et al (2000) FEBS Letters, 471, pp.147). Thus, it appears that the mutant Int can be recombined without the need to form a defined nucleoprotein complex, such as an integrant assembled on attP. This raises the question of the functional role of the DNA arm binding site in recombination. They are located in at least one att site partner (partner) used so far.

To investigate this problem, we performed intermolecular recombination with paired substrate vectors containing attB or attP in various combinations (FIG. 3A). After cotransfection with the Int expression vector for 48h, the proportion of recombinantly produced GFP-expressing cells was measured by FACS. Transfection efficiency was always higher than 90% (data not shown). The results of 3 trials showed that intermolecular recombination between pairs of attP was as efficient as recombination between attB and attP (fig. 3B). However, to a large extent, only Int-h/218 utilizes pairs of attB sites as substrates. The efficiency of this reaction is reduced on average by a factor of about 4 compared to the reaction between attP and attP or attB and attP (fig. 3B). Thus, the proportion of GFP-expressing cells produced by recombination between the two attB sites was reduced to a level of 4-5%. These results demonstrate that the presence of the armed sequence in the att site is not essential for the recombination reaction by Int-h/218, but significantly stimulates this reaction. This stimulatory effect was even more pronounced (about 8-fold) when Int-h was used. In addition, the residual recombination activity observed with wild-type Int appears to be highly dependent on the presence of the arm binding site.

Example 3: stimulation of recombination by wild-type Int by transfected IHF protein

Efficient integrated recombination catalyzed by wild-type Int in vitro and in e.coli requires the protein cofactors IHF and attP supercoils. The apparent absence of either cofactor in mammalian cells has led us to investigate whether the residual recombinant activity of wild-type Int is increased if purified IHF pre-cultured with supercoiled substrate is co-introduced into HeLa cells. To verify this possibility, wild-type Int or Int-h expression vectors were first introduced. After 3 to 4h of electroporation, the substrate for intramolecular or intermolecular recombination is incubated with or without purified IHF. The protein-DNA complex was then transfected with Fugene as well as a protein-free control sample (fig. 4A). After another 48h the ratio of GFP expressing proteins was compared.

The results of 3 experiments showed that intramolecular recombination by wild-type Int increased on average to 5-fold due to the presence of IHF. The proportion of GFP positive cells increased, for example, in one experiment, from about 1% in the absence of IHF to 6% in the presence of IHF. The stimulatory effect on intermolecular recombination was also significant, but small (about 3-fold). After 48h of transfection, the stimulatory effect was directed only to wild-type Int, since Int-h activity was not affected. Importantly, the control group showed that the presence of IHF protein also did not affect transfection efficiency (data not shown).

Example 4: improved protein expression system based on sequence-specific recombination of target genes

CHO-DG44 cells were stably transfected with linear primary plasmid DNA expressing the fluorescent protein ZsGreen1 from Zoanthus sp (Clontech Laboratories inc., Palo Alto, CA, u.s.a.) and the antibiotic resistance gene neomycin phosphotransferase (fig. 5). In addition, attB or attP recombination sequences (natural or modified sequences or derivatives thereof) are placed between the fluorescent protein gene and its promoter. The first plasmid DNA was linearized with a restriction enzyme having a single restriction site outside the transcription units of the two selectable markers and introduced into the genome of CHO-DG44 by random integration. Cells that successfully stably and randomly integrated the first plasmid DNA were positively selected by culturing in the presence of the antibiotic G418. In a heterogeneous pool of stable transfectants, cells with high transcriptional activity at the first plasmid DNA integration site can be isolated by mere Fluorescence Activated Cell Sorting (FACS) based on the expression level of the introduced fluorescent protein ZsGreen 1. Cells with the highest ZsGreen1 fluorescence were picked and placed as single cells into wells of a 96-well plate. The resulting cells were subcloned and examined for integration of a single plasmid sequence at a single chromosomal site by restriction enzyme mapping in Southern blot analysis. For the latter, the genomic DNA of the cell subclones was digested with restriction enzymes having one and more restriction sites, respectively, which were absent from the introduced first plasmid DNA, electrophoresed on 0.8% agarose gels and transferred onto positively charged nylon membranes (Amersham Biosciences, Freiburg, Germany). Hybridization was carried out overnight in a 65 ℃ hybridization oven according to Gene Images random priming module (Amersham Biosciences) using a random priming FITC-dUTP labeled probe consisting of the ZsGreen1 Gene. Candidate subclones with single copy inserts were then tested for performance in simulated production batch cultures in a small scale bioreactor. In addition to the high expression levels monitored by measuring the fluorescence of ZsGreen1, other important parameters such as high viability at high cell densities, metabolic and reproductive performance are also considered during all production phases. Suitable host cells with integrated first att recombination sequences are thus identified. To generate a production cell line for the production of biopharmaceuticals by sequence-specific recombination, this host cell is transfected with a second plasmid DNA (see FIG. 5) containing a promoterless dihydrofolate reductase gene following the attB or attP recombination sequences (natural or modified or derivatives thereof) and the complete transcription unit capable of expressing the gene of interest, such as sICAM (soluble intermolecular adhesion molecule 1) for the treatment of the common cold or human monocyte chemoinducible protein-1 (MCP-1). In addition, a vector pCMVSSInt-h/218 expressing a mutant (modified) lambda phage integrase was co-transfected. After transfection, transient expression of Int-h/218 is sufficient for sequence-specific intermolecular recombination between the first att recombination site (attP or attB) at the preferred transcriptionally active locus in the host cell genome and the second att recombination site (attP or attB) on the introduced second DNA plasmid. To screen for cells that have undergone sequence-specific recombination between attP and attP, attP and attB or attB and attB (depending on the recombination sequences on the first and second DNA plasmids), the transfected cells were transferred to CHO-S-SFMII medium that was not supplemented with hypoxanthine and thymidine and cultured. The dhfr selectable marker gene is expressed sufficiently by placing the promoterless dhfr-marker gene and the upstream att recombination site of the second DNA plasmid under the control of the promoter sequence of the ZsGreen1 gene and the downstream att recombination site by recombination so that only the correct orientation results in the cells surviving the screen. Meanwhile, the functional expression cassette of the ZsGreen1 gene is interrupted, and a promoterless ZsGreen1 gene is left. The cells no longer express the fluorescent protein. Non-fluorescent cells are identified and isolated using FACS, which provides a means of detecting cells that produce the protein of interest. In addition, sequence-specific integration was verified by Southern blotting and PCR analysis with primers located in the sequences flanking the att sites, before and after site-specific recombination, followed by DNA sequencing. Expression of the target protein, sICAM or MCP-1 was analyzed by ELISA.

The use of DHFR as a marker gene for the generation of producer cell lines provides not only the advantage of positive selection, but also the possibility of increasing the productivity of the cells by methotrexate-induced DHFR gene amplification. This was achieved by supplementing the CHO-S-SFMII medium without hypoxanthine/thymidine with increasing amounts of methotrexate.

Claims (17)

1. An in vitro or ex vivo method of expressing at least one target gene encoding one or more desired therapeutic proteins, polypeptides, fragments thereof, peptides, and/or antisense RNAs in a eukaryotic cell other than a human oocyte and embryonic stem cell, the method comprising

a) Introducing a first DNA comprising an attB, attP, attL or attR sequence into a cell;

b) introducing into the cell a second DNA comprising an attB, attP, attL or attR sequence and at least one target gene encoding a therapeutic protein;

c) contacting said cell with a lambda phage integrase Int;

d) sequence-specific recombination by bacteriophage lambda integrase Int, wherein the second DNA is integrated into the first DNA; and

e) culturing the cell under conditions that allow expression of the gene of interest.

2. The method of claim 1, wherein when said first DNA sequence comprises an attB sequence, said second sequence comprises an attB, attL or attR sequence, or when said first DNA sequence comprises an attP sequence, said second sequence comprises an attP, attL or attR sequence, or when said first DNA sequence comprises an attL sequence, said second sequence comprises an attB, attP or attL sequence, or when said first DNA sequence comprises an attR sequence, said second sequence comprises an attB, attP or attR sequence.

3. The method of claim 1 or 2, wherein the first DNA has been integrated into the genome, artificial chromosome or minichromosome or episomal element of the cell prior to the second DNA being introduced into the host cell.

4. The method of claim 3, wherein the first DNA is integrated into the genome of the host cell.

5. The method of any one of claims 1 to 4, wherein the desired therapeutic protein, polypeptide, fragment thereof, peptide, and/or antisense RNA is isolated from the host cell or cell culture medium.

6. The method of any of the preceding claims, wherein said sequence-specific recombination is performed by a modified Int and one or more cofactors selected from the group consisting of XIS, FIS and/or IHF, wherein said modified Int is Int-h or Int-h/218.

7. Method according to one of claims 1 to 5, wherein said sequence-specific recombination is performed by a modified Int, wherein said modified Int is Int-h or Int-h/218.

8. The method of claim 6 or 7, wherein Int, Int-h or Int-h/218, XIS, FIS and/or IHF is added to the cell in purified form or is co-expressed by said host cell subjected to sequence specific recombination.

9. Method according to any of the preceding claims, wherein additionally a third or a third and a fourth DNA sequence comprising a modified Int gene, respectively, or a modified Int gene and one or more cofactor genes selected from the group of XIS gene, FIS gene and/or IHF gene is introduced into the cell, wherein said modified Int is Int-h or Int-h/218.

10. The method of any of the preceding claims, wherein neither XIS, FIS nor IHF is required when sequence-specific recombination is performed with a modified Int, wherein the modified Int is Int-h or Int-h/218.

11. The method of any of the preceding claims, wherein the first and/or second recombinant sequence further comprises a nucleic acid encoding a therapeutic polypeptide of interest.

12. The method of any of the preceding claims, wherein the therapeutic polypeptide of interest is an antibody, hormone or growth factor.

13. The method of any one of the preceding claims, wherein the host cell is a mammalian cell.

14. The method of claim 13, wherein the mammalian cell is a rodent cell.

15. The method of claim 14, wherein the rodent cell is a mouse or hamster cell.

16. The method of claim 15, wherein the hamster cell is BHK or CHO cell and the mouse cell is a murine myeloma cell.

17. The method of claim 16, wherein the murine myeloma cells are NS0 and Sp2/0 cells.