The method of embedding the desired dna into the genome of mammalian cells and vector system for its implementation

 

The invention relates to the field of genetic engineering and can be used in the biotechnology industry. The method of integration of DNA of interest into the site of the genome of mammalian cells, characterized by high transcriptional activity. The site mentioned previously labeled by introducing into the cell a specially designed plasmid (“marker”), including (a) a fragment of heterologous with respect to the genome of the cell's DNA, which after integration into the genome provides a unique site for homologous recombination; b) a DNA fragment encoding a portion of the first selective marker, and C) at least one marker DNA sequence, allowing for selection of cells that have successfully passed integration “marker” plasmids. Selected in the first stage cells then transform the second (“target”) plasmid comprising (a) a DNA fragment having sufficient to allow recombination homology with a unique website and “marker” plasmid; b) a DNA fragment encoding the second portion of the first selective marker co-expression with the element (b) “marker” plasmids provides a synthesis of the complete marker protein and DNA, Colette, in which DNA targeted plasmids and therefore “need” DNA, was included in the genomic DNA. Offered a kit for implementing the method, containing at least “marker” and “target” plasmids. The use of the invention provides a high level of expression with any of the recombinant proteins. 2 C. and 39 C.p. f-crystals, 10 ill., 5 table.

The scope to which the invention relates

The present application relates to a method for the directed integration of the desired exogenous DNA in a specific region of the genome of mammalian cells. More specifically, the present invention describes a new method for the identification of transcriptionally active target site ("hot spots") in the genome of the mammal, and embed the desired DNA into this site by homologous recombination. The present invention also, but not necessarily, refers to the ability of gene amplification of the desired DNA into this site by co-integration amplifierarava breeding marker such as DHFR, in combination with exogenous DNA. In addition, the present invention describes the construction of new vectors that are suitable for implementing the foregoing methods, as well as the production of these methods to the ke".

Background of invention

The technique of expression of recombinant proteins in prokaryotic and eukaryotic microorganisms are well developed. From the point of view of the production of proteins, mammalian cells have significant advantages compared with bacteria or yeast due to its ability to correct the Assembly, glycosylation and post-transcriptional modification recombinante expressed proteins. After transfection into cells of the host, the recombinant expression constructs can be stored in the form of extrachromosomal elements, or they can be integrated into the genome of the host cell. Generation of stably transfected cell lines mammals usually involves embedding the above construction, that is, the DNA construct encoding the desired gene along with the gene of resistance to the drug (dominant selective marker) introducing into the host cell; and then these cells are grown in the presence of drugs, allowing for selection of cells containing a successfully integrated exogenous DNA. In many cases, the desired gene is attached to a selective marker of resistance to the drug is gene encoding dihydrofolate-reductase (DHFR). Growing cells in the presence of methotrexate, a competitive inhibitor of DHFR leads to an increased production of DHFR by amplification of DHFR gene. As amplification also flanking DNA segments that carry out co-amplification of the gene attached to DHFR, in transfected cell lines can lead to increased production of the protein, which will provide a high level of expression of the desired gene.

Although this approach gives good results, however, described the system creates a number of problems associated with the random nature of the events integration. These problems arise from the fact that the levels of expression largely influenced by local effects of the genetic environment in the gene locus, and these phenomena have been well described in the literature, and are usually called "effects of provisions" (see, for example, Al-Shawi et al., Mol. Cell. Biol., 10:1192-1198 (1990); Yoshimura et al., Mol. Cell. Biol., 7:1296-1299 (1987)). Because the vast majority of mammalian DNA is transcriptionally inactive state, the methods of random integration does not provide control of the transcriptional state of the integrated DNA. Therefore, it may shirokoekrannyy exogenous DNA in transcriptionally inactive or "silent" areas of the genome will lead to a small expression or even its absence. In contrast, the integration into a transcriptionally active site can lead to high levels of expression.

Therefore, if the aim of the study is to obtain high levels of gene expression, the achievement of which is usually the desired result of genetic engineering methods, for detection of the high-producing clone, basically, you need to skanirovat a large number of transfectants. In addition, in some cases, random integration of exogenous DNA into the genome can cause destruction of important cellular genes that leads to a change in phenotype. Because of these factors, generating vysokoagressivnyh stable cell lines mammals can become complex and time consuming process.

Recently in our laboratory described the use of DNA vectors containing translational weakened dominant selective marker for gene expression mammals. (This was described in the patent application U.S. reg. No. 08/147696, filed November 3, 1993 and recently recognized as patentable).

These vectors contain translational weakened gene neomycin-phosphotransferase (peo) as the dominant breeding marker, artificially constructed so that it soavneniju with standard expressing vectors mammals, the use of these vectors as expressibly structures significantly reduces the total number produced colonies that are resistant to the drug, thereby facilitating the screening procedure. In addition, a significant percentage of clones obtained with the use of this system constitutes visokoaktivnie clones. These results, apparently due to modifications made in breeding marker peo. Due to translational attenuation neo gene, transfected cells will not produce protein neo at a level sufficient for the survival of cells resistant to the drug, thereby, reducing the total number of colonies resistant to this drug. In addition, a higher percentage of surviving clones will contain expressing vector-integrated sites in the genome, where basal levels of transcription are quite high, which will lead to overproduction neo gene, and thereby avoids damage to the neo gene in these cells. In line with this, the desired genes attached to the neo gene will be subject to a similar increase in the level of transcription. The same benefits can be access from the synthesis of functional neo gene, which, in turn, depends on the proper and efficient splicing of introns neo. Moreover, these criteria are, in all probability, are satisfactory in the case if the DNA of the vector integrated in an area that is already highly transcriptionally active.

After integration of the vector in a transcriptionally active region, the gene amplification is carried out by selection for DHFR gene. Using this system it is possible to obtain clones selected using low levels of methotrexate (50 nm) containing a small amount (< 10) copies of the vector, which secretes high levels of the protein (> 55 PG/cell/day). In addition, this can be achieved in a relatively short period of time. However, such amplification is carried out with varying degrees of success. Some of transcriptionally active sites cannot be amplified, and therefore the frequency and the degree of amplification of a specific site is unpredictable.

In General, the use of these transcription weakened vectors represents a greatly improved method compared with other methods of random integration. However, as already discussed, the lack of control in respect of the site of inteha, is a method of targeted gene transfer, resulting in targeted integration of exogenous DNA at the specific locus in the genome of the host. Exogenous DNA is integrated by homologous recombination occurs between DNA sequences in expressing vector and the corresponding homologous sequences in the genome. However, in yeast and other fungal pathogens, this type of recombination occurs in nature with high frequency, whereas in higher eukaryotic organisms, this type of recombination is an extremely rare event. It was found that in mammalian cells, the frequency of homologous recombination in relation to non-homologous recombination (random integration) is in the range from 1/100 to 1/5000 (see, e.g., Capecchi, Science, 244:1288-1292 (1989); Morrow & Kucherlapati, Curr. Op. Biotech., 4:577-582 (1993)).

In one of the earliest works described homologous recombination in mammalian cells, containing an artificial system created in murine fibroblasts (Thomas et al., Cell 44:419-428 (1986)). Was established cell line containing a mutated nonfunctional version of the neo gene integrated into the host genome, and then it was introduced by the target case is t to occur only when directed gene transfer. Homologous recombinants were identified by selection of cells for resistance to G418, and confirmed by analysis of genomic DNA extracted from drug-resistant clones.

Recently it was reported about the use of homologous recombination to replace the genes of heavy and light chain immunoglobulin in the endogenous locus in antibody-secreting cells (U.S. patent No. 5202238, Fell et al., (1993)). However, this particular method is not widely adopted because it is limited to the production of immunoglobulins in cells that Express endogenous immunoglobulins, for example, in b cells and myeloma cells. This expression is also limited by the levels adenocarinoma gene due to the fact that after homologous recombination does not occur to amplification. In addition, this method is complicated by the fact that for the production of a functional immunoglobulin requires two separate events integration; one for the light chain gene, and then one for the heavy chain gene.

An additional example of a system of this type is described for cells, NS/0, where the recombinant antibodies expressed by homologous recombination at the locus 2A gamma immunoglobulin (Hollis et al. International patent application # PCT/IB95 (00014) ). Orinoko of integrant. However, as in the example above, the expression is limited to this level because amplificatory gene is not co-integrated in this system. Other researchers also reported the aberrant glycosylation of recombinant proteins expressed in cells, NS/0 (see, for example, Flesher et al., Biotech. and Bioeng., 48:399-407 (1995)), thus limiting the application of this approach.

Recently received a recombination system cre-loxP, derived from bacteriophage P1 and used for targeted gene transfer in eukaryotic cells. In particular, was described site-specific integration of exogenous DNA into the genome of cells of the Chinese hamster ovary (Cho) using recombinases, she and series Oh-containing vectors. (Fukushige & Sauer, Proc. Natl. Acad. Sci., USA, 89:7905-7909 (1992) ). This system is attractive because it provides a replicable expression in the same position on the chromosome. However, it was not made any attempts to identify the site of the chromosome at which the gene expression is optimal, and, as in the previous example, the expression in this system is limited odnokorennye levels. And in this case there are complications due to the fact that it is necessary to provide expressional recombination between the introduced DNA sequence and its endogenous chromosomal locus for the development of effective methods of genetic manipulation in mammalian cells, as well as in yeast cells (see, e.g., Bradley et al., Meth. Enzymol., 223:855-879 (1993); Capecchi, Science, 244:1288-1292 (1989); Rothstein et al., Meth. Enzymol., 194; 281-301 (1993)). To date, studies relevant to the target gene transfer in most mammalian cells, were aimed at the destruction of the gene ("knock-out") or site-directed mutagenesis of the loci selected target genes in stem cells of mouse embryos (ES). The creation of these mouse models "knockout" has enabled scientists to identify specific structural and functional outputs and to assess the biological value of the myriads of mouse genes. This area of study is also important from the point of view of possible applications in gene therapy.

Recently, Celltech (Kent, U. K.) described vectors, which were aimed at transcriptionally active sites in cells N30, does not require amplification of the gene (Peakmann et al., Hum. Antibod. Hybridomas, 5:65-74 (1994)). However, in this work it was reported that the levels of secretion of immunoglobulins in these reamplification cells can exceed 20 PG/cell/day, and in amplified cells SNO can be received levels that reach 100 PG/cell/day (ibid.).

It would be highly desirable to develop a system of targeted gene transfer, catname, aware that he is transcriptionally active. In addition, it would also be desirable that such a system of targeted gene transfer could facilitate co-amplification of the integrated DNA after its integration. The design of such a system would allow for replicable and highly efficient expression of any interest cloned gene in a cell of a mammal, and certainly would be of great interest to researchers.

In this application we have described a new expressing the system of mammals obtained on the basis of homologous recombination occurring between two artificial substrates contained in two different vectors. In particular, in this system there are two new expressing vector mammals, entitled "marking vector and the target vector transfer".

Basically marking vector provides identification and marks the site of the genome of the mammal, which is transcriptionally active, i.e. the site where the levels of gene expression are high. This website can be considered as a "hot spot" in the genome. After integrating this marking vector, consider expressing siginal recombination, what is happening between DNA sequences that are common to these two vectors. This system has significant advantages compared with other systems of homologous recombination.

Unlike most other systems homologous recombination used in mammalian cells, this system does not give the background level. Therefore, cells that are only random integration of the vector, do not pass screening. Thus, the desired gene is cloned into a plasmid target for migration, is expressed at high levels from the marked "hot spots". Accordingly, the method of gene expression allows mainly or completely solve the problems associated with random integration, described in detail above. Moreover, this system provides a replicable and highly efficient expression of any of the recombinant protein in the same transcriptional active site of the genome of the mammal. In addition, gene amplification can be carried out in this specific transcriptional active site by enabling amplifierarava dominant selective marker (e.g., DHFR) as part of the marking vector.

The purpose of the present invention

That the DNA into a specific site of mammalian cells.

A more specific aim of the present invention is to develop a new way of targeted transfer the desired DNA into a specific site of mammalian cells by homologous recombination.

Another specific aim of the present invention is to obtain new vectors for site-specific integration of the desired DNA into the cell of a mammal.

Another objective of the present invention to provide a new cell lines of the mammal containing the desired DNA is integrated into a pre-defined website, which provides a high level of expression.

A more specific aim of the present invention is to develop a new way to achieve site-specific integration of the desired DNA into the cells of the Chinese hamster ovary (Cho).

Another more specific objective of the present invention is to develop a new way of integrating immunoglobulin genes or other genes in mammalian cells in pre-defined chromosomal sites, which provide a high level of expression.

Another specific aim of the present invention is to obtain new vectors or combinations of vectors suitable for the integration of genes immunol is I.

Another objective of the present invention to provide cell lines mammals, containing immunoglobulin genes integrated into pre-defined sites, which provide a high level of expression.

The more specific aim of the present invention is to develop a new way of integration of immunoglobulin genes in cells of Cho, which provide a high level of expression, as well as obtain new vectors or combinations of vectors suitable for such integration immunoglobulin genes in cells SNO.

In addition, the specific objective of the present invention to provide a new cell lines Cho, which contain immunoglobulin genes integrated into pre-defined sites, providing a high level of expression, and which were amplified by selection with the use of methotrexate for the secretion of large quantities of functional immunoglobulins.

Fig.1 depicts a map marking the plasmids of the present invention, named Desmond. This plasmid is shown in circular form (1A) and the linearized version used for transfection (1b).

Fig.2 (a) depicts the plasmid map of the target of transfer, called "Molly". Displayed is(b) depicts a linearized version of Molly after its hydrolysis by restrictase and kpni restriction sites PacI. This linearized form used for transfection.

Fig.3 illustrates a possible comparison of the primary integrated into the SNO-genome sequences Desmond and the inserted sequences of the plasmid target transfer Molly. Also presented one of the possible structures posledovatelnoy Molly integrated sequence Desmond after homologous recombination.

Fig.4 shows southern analysis odnoletnih clones Desmond. Samples were:

Lane 1: marker sizeHindIII-DNA

Track 2; clone Desmond 10F3

Lane 3: clone Desmond 10C12

Lane 4: clone Desmond 15C9

Track 5: the clone Desmond 14B5

Track 6: the clone Desmond W.

Fig.5 shows Northern analysis odnoletnih clones Desmond. Samples were: Panel A: Northern clones probed CAD and DHFR probes, as shown in the figure. Panel: duplicate Northern clones probed CAD and HisD probes, as shown RNA samples loaded on Panels a and b, were as follows: lane 1: clone 9B2; lane 2: clone 10C12; lane 3: clone V; lane 4: clone 15C9; lane 5: control RNA from cells SNO, transfected HisD and DHFR-containing plasmid; lane 6; nitrostilbene SNO.

Fig.6 shows a southern analysis of clones D/chr/955.gif">HindIII-DNA; lane 2: 20F4; lane 3: 5F9; lane 4: S; track 5: 24G2; lane 6: E; track 7: S; lane 8: 29F9; lane 9: 39G11; track 10: 42F9; track 11: 50G10; track 12: plasmid DNA Molly, linearized with the enzyme BgIII (upper band) and cut by enzymes BgIII and kpni restriction sites (lower band); lane 13: atransferrinemia Desmond.

Fig.7A-7C represent the List of sequences for Desmond.

Fig.8A-8I are the List of sequences to plasmids Molly, containing the gene of an antibody against CD20.

Fig.9 is a map of plasmid target transfer, "Mandy", which shows the genes that encode the immunoglobulin against CD23 and the expression of which is described in Example 5.

Fig.10A-10N is a list of sequences of plasmids "Mandy", containing the genes for immunoglobulin against CD23 described in Example 5.

Detailed description of the invention

The present invention relates to a new method of integrating the desired exogenous DNA into the target site of the genome of mammalian cells by homologous recombination. The present invention also relates to new vectors to achieve site-specific integration of DNA into the target site of the genome of mammalian cells.

More specifically, the method cloned the TCI "marker plasmid", contains a unique sequence that is alien to these genome of mammalian cells, with subsequent transfection "target plasmid transfer containing a sequence that provides for homologous recombination with a unique sequence contained in the marker plasmid, and in addition, includes the desired DNA, which must be integrated into the cell of a mammal. Basically, integrated DNA will encode the desired protein, such as an immunoglobulin or another glycoprotein, the secretory cells of the mammal.

In this system, homologous recombination, as a dominant selective marker used gene neomycin-phosphotransferase. This particular marker was used based on the following previously published data:

(i) the demonstrated ability of this marker to directed migration and preserve the function of the mutated version of the gene neo (as mentioned earlier); and

(ii) our development of translational weakened expressing vectors, in which the neo gene was artificially created in the form of two exons with the desired gene integrated into the intron, where these exons neo correctly spiceroads and trantest to G418. In this application, the neo gene was divided into three exons. The third exon neo is on the "marker" plasmid and integrated into the genome of the host cell after integration marker plasmids in mammalian cells. Exons 1 and 2 are present on the target plasmid transfer, and separated by an intron, which was cloned at least one desired gene. Homologous recombination vector, the target transfer with integrated marker vector leads to the correct splicing of all three exons of the gene neo, and thus to the expression of functional protein neo (as determined by selection of the resistance of the colonies to G418). Before constructing considered expressing the system, we conducted an experimental test of the functionality of that thrice splanirovannaya neo-constructs in mammalian cells. The results of this control experiment showed that all three neo-exons were correctly splanirovany that gave reason to assume the technical feasibility of the present invention.

Although the present invention is illustrated using the neo gene, and more specifically neo gene with triple splitting, however, the General methodology should be effective using the other to the creation of advantages in comparison with standard methods of gene expression, including as a way of random integration, and targeted gene transfer. In particular, the present invention relates to a method, which allows replicable site-specific integration of the desired DNA in a transcriptionally active domain of mammalian cells. Moreover, because the method allows to introduce an artificial area "homology", which acts as a unique substrate for homologous recombination, and embed the desired DNA, for the effective implementation of the present invention does not require this cell endogenous contained or expressed specific DNA.

Thus, this method is genetically applicable to all mammalian cells and can be used for the expression of any type of recombinant protein.

Use three times splanirovano selective marker, for example, consider three splanirovannaya neo-design, gives the assurance that all biogas produced G418-resistant colonies will be formed following the events of homologous recombination (random integrant will not produce a functional neo gene, and therefore, they will not survive when S-selection). Thus, the CSOs, it is obvious that the frequency of additional random integration in the cell, which is exposed to the events of homologous recombination is low.

Based on the above, it is clear that a significant advantage of the present invention is that it allows mainly to reduce the number of colonies that you want to skanirovat to identify high-yielding clones, i.e., cell lines containing the desired DNA, which secretes the corresponding protein at high levels. On average, clones containing integrated the desired DNA can be identified by screening from about 5 to 20 colonies (compared to the few thousand that must be skanirovaniya when using the standard technique of random integration, or a few hundred, which should be skanirovaniya using the previously described vectors with intron-insertion). In addition, because the site of integration was pre-selected and contains transcriptionally active domain, all exogenous DNA, expressed in this site should produce comparable, i.e., high levels of the desired protein.

In addition, the present invention is preferable in that it obegin directed to this site by homologous recombination, the present invention allows to Express the gene, even more amplified by the amplification of genes. In this regard, in the literature it was reported that different genomic sites have different ability to gene amplification (Meinkoth et al, Mol. Cell. Biol., 7:1415-1424 (1987)). Therefore, this technique is preferred since it allows you to place the desired gene into a specific site that is both transcriptionally active and easily amplificare. Therefore, this technique should lead to a significant reduction in the time required for making such highly efficient producers.

In particular, standard methods for the design of highly expressing cell lines mammals can take from 6 to 9 months, whereas the present invention allows to select the clones, on average, only for 3-6 months. This is due to the fact that to obtain satisfactory levels of expression of genes selected in the standard way clones usually must be at least three cycles of amplification of the gene resistant to the drug. Because homologous produced clones were generated from pre-selected sa require fewer cycles of amplification.

In addition, the present invention provides a replicable selection of highly productive clones in which the vector is integrated with a low number of copies, usually with a single copy. It is an advantage that will improve the stability of clones and avoid other possible unwanted side effects associated with a high number of copies. As described above, in this system, homologous recombination is used a combination of marker plasmids and plasmid target transfer", which are described in more detail below.

"Marker plasmid, which is used for marking and identification of transcription "hot spots" that contains at least the following sequence:

(i) a region of DNA that is heterologous or unique to the genome of mammalian cells, which functions as a source of homology, providing homologous recombination (DNA contained in the second plasmid target of the transfer). More specifically, this unique region of DNA (i) mainly contains bacterial, viral, yeast, or other synthetic DNA, which is normally not present in the genome of normal cells of the mammal and which, moreover, does not have znachitelnie mammal. Basically, this sequence must sufficiently different from the DNA of a mammal that is not exposed to significant recombination with the genome of the host cell by homologous recombination. As some other researchers have noted an increase in the frequency of the target recombination with increasing size of the region of homology (Capecchi, Science, 244:1288-1292 (1989)), the size of such a unique DNA will mainly be at least about 2-10 thousand base pairs or greater, and more preferably at least about 10, etc., O.

The upper limit of the amount of unique DNA that serves as a site for homologous recombination with the sequence of the second target vector, mainly due to the possible limits of stability (if the DNA is too large, it cannot be easily integrated into the chromosome, which makes it difficult to work with very large DNA).

(ii) a DNA comprising a fragment of the selective marker DNA, usually exon dominant selective marker gene. The main distinguishing feature of this DNA is that it does not encode a functional breeding marker protein, although it is expressed together with the sequence, the soda is Chernogo gene (which is not present in the plasmid directional transfer"). Mostly functional selective marker should be produced only when homologous recombination (resulting in binding and expression of this marker DNA sequence(i) together with part (s) of the slice selective marker DNA, which is contained in the target plasmid).

As indicated, the present invention illustrates the use of gene neomycin-phosphotransferase as a dominant selective marker, which "splits" into two vectors. However, it may be appropriate also other selective markers, such as gene histidinol-dehydrogenase of Salmonella, gene hygromycin-phosphotransferase, gene thymidine kinase of herpes simplex virus, gene adenosine-deaminase, the gene for glutamine synthetase gene gipoksantin-geoinformational-transferase.

(iii) DNA encoding a functional selective marker protein, where the specified selective marker is different from the selective marker DNA (ii). This selective marker provides a successful selection of mammalian cells, where the marker plasmid was successfully integrated into the cellular DNA. More preferably, this marker plasmid contained two such dominant selective marker DNA is the welts can be selected using various selective agents and in addition, can be selected cells that contain a complete selective vector. In addition, one marker can be amplificare marker to facilitate amplification of the gene, as previously discussed. Can be used any of a dominant selective marker listed in (ii), and can also be used with other markers, is well known to specialists.

Moreover, the marker plasmid may, but not necessarily, further comprise a rare restriction site for the endonuclease. This option is potentially desirable because in this case can be facilitated cleavage. Such a rare restriction site, if present, must be located almost in the middle of a unique region, which serves as a substrate for homologous recombination. Preferably, such a sequence comprised of at least about 12 nucleotides. It was reported that the introduction of double-strand break using a similar technique increases the frequency of homologous recombination (Choulika et al., Mol. Cell Biol., 15:1968-1973 (1995)). However, the presence of such a sequence is not critical.

"The target plasmid transfer" includes at least the following posledovateley sufficient homology or identity with the field, where this DNA can be integrated by homologous recombination with a unique region (i) in the marker plasmid. Suitable types of DNA described above in the description of the unique region of DNA (1) in the marker plasmid.

(2) the remaining exons dominant selective marker, of which one exon is included as (ii) in the marker plasmid as defined above. The main feature of this DNA fragment is that it enables the production of functional (selective) marker protein only in the case when the target plasmid is integrated by homologous recombination (where this recombination leads to the binding of DNA with another slice selective marker DNA present in the marker plasmid), and that it contributes to the embedding of the desired exogenous DNA. Basically this DNA will include the remaining exons selective marker of DNA that are separated by an intron. For example, this DNA may contain the first two exons of the gene neo, and marker plasmid may contain the third exon (third behind neo).

(3) Target plasmid will contain the desired DNA, such as DNA encoding the desired polypeptide and preferably integrated in the slice selective the selective marker exons DNA. This helps to ensure that the desired DNA is integrated into the case, if there is a homologous recombination of the target plasmid and marker plasmids. This intron may be natural or it can be constructed in a dominant selective marker DNA fragment.

This DNA will encode any desired protein, preferably a protein with pharmaceutical or other desirable properties. Basically, most of the DNA will encode a protein of a mammal, and in the following examples immunoglobulin or immunoadhesin. However, the present invention is in no way limited to the production of immunoglobulins.

As discussed previously, the tool of the present invention can be applied to any cell of a mammal, because its effectiveness does not require the presence of any specific sequence or sequences of the mammal. Basically such mammalian cells are cells that are commonly used for protein expression, for example cells of Cho, myeloma cells, COS cells, cell KSS cells Sp2/0 cells, NIH T and HeLa. In the examples below, used cells SNO. Their advantage lies in their ability barb in culture, as well as in their ability to Express mammalian proteins, such as immunoglobulins, in biologically active form.

In addition, cells SNO were selected mainly because of the previous use of these cells by the authors of the present invention for the expression of immunoglobulins in (using translational weakened vector containing a dominant selective marker, and described earlier). Thus, our laboratory has considerable experience in the use of such cells for expression. However, from the examples below, there is every reason to expect that similar results will be obtained using other mammalian cells.

Basically the transformation or transfection of mammalian cells in accordance with the present invention can be carried out by standard methods. Therefore, for a better understanding of the present invention, the following examples describe illustrative design vectors and their use for the production of integrants.

Example 1

Designing and obtaining marker plasmid DNA vectors and plasmid DNA vectors for the target transfer

Marker plasmid, designated in this API transcription cluster containing the promoter of the mouse beta-globin 5'side with respect to the originating site of DHFR, and the polyadenylation signal of bovine growth hormone 3'-side relative to the stop codon. Transcriptional cluster DHFR was isolated from TSAE expressing vector created previously in this laboratory (Newman et al., 1992, Biotechnology, 10:1455-1460).

(b) Gene-galactosidase of E. coli, commercially available gene supplied by Promega as a control vector pSV--galactosidase, catalog # E.

(c) Baculovirus DNA, commercially available DNA supplied from Clontech as rakrak, cat. # 6145-1.

(d) cluster, including the promoter and enhancer elements from the CMV and SV40 virus. This cluster was constructed by the PCR using the derived expressing vector TCAE (Reff et al., Blood 83:435-445 (1994)). This enhancer cluster was introduced in baculovirus sequence, which was first modified by embedding the multiple cloning site.

(e) Gene GUS (glucuronidase) E. coli, commercially available gene supplied by Clontech in the form RW, cat. # 6017-1.

(f) luciferase Gene of the Firefly, commercially available gene supplied by Promega in pGEM-Luc who Donahue et al., Gene, 18:47-59 (1982), and was then introduced in the transcriptional cluster containing the promoter of the mouse beta-globin 5'side with respect to gene, and the SV40 polyadenylation signal 3'-side in relation to gene.

DNA elements described in (a)-(g) were combined in a plasmid backbone, derived from pBR, producing a continuous DNA fragment 7.7, etc., of O., indicated in the accompanying illustrations as "homologous" ("homology"). The term "homologous" in this case refers to DNA sequences that are not part of the mammalian genome and are used to stimulate homologous recombination between trapezitinae plasmids that share homologous DNA sequences.

(h) Gene neomycin-phosphotransferase from TN5 (Davis & Smith, Ann. Rev. Micro., 32:469-518 (1978)). Full neo gene was subcloned into pBluescript SK-(Stratagene catalog # 212205) to facilitate genetic manipulation. Then the synthetic linker was built in the unique Pstl site spanning codons neo gene for amino acids 51 and 52. This linker encodes the desired DNA elements to create artificial donor site splicing, introns and acceptor site of splicing in the gene neo, which created two separate exon, designated in the present description as neo-exon 1 is Odon protein A. This intron was also created Notl site cloning.

Neo-exon 2 was also subdivided into neo-exons 2 and 3. This was achieved as follows: there was constructed a series of PCR primers to amplify the region of DNA that encodes a neo-exon 1, intron and the first 111 2/3 amino acids of exon 2. 3'-RAC-primer led to the introduction of a new 5'-site of splicing, which was located directly behind the second nucleotide of the codon for amino acid 111 in exon 2, and thereby, generating a new smaller exon 2. Then, this DNA fragment encoding is now similar exon 1, intron and a new exon 2, was subcloned and expanded in the vector, derived from pBR. The rest of the initial exon 2 was used as the matrix for another cycle PCR amplification, which was generated by "exon 3" 5'-primer for this round of amplification has introduced a new acceptor site of splicing with the 5'-side of the new created exon 3, that is, before the end nucleotide of the codon for amino acid 111. Received 3 exon gene neo encode the following elements: exon 1 - the first 51 amino acids neo gene; exon 2 - the following 111 2/3 amino acids, and exon 3 - final 91 1/3 amino acids plus the termination codon of the neo gene transcription.

Neo-exon 3 was introduced vmesto "Molly". Notl site clone, created in the intron between exons 1 and 2 was used in the subsequent stages of cloning to embed the desired genes into the target plasmid transfer.

Was also constructed a second plasmid target transfer "Mandy". This plasmid is almost identical to the plasmid "Molly" (changed some restriction sites on the vector) except that plasmid "Molly" were inactivated initially present HisD genes and DHFR. These changes were made because the cell line containing plasmid Desmond, can no longer be cultured in the presence of histidinol, and therefore, obviously, it is not necessary to include a second copy of the HisD gene. In addition, the DHFR gene was inactivated to ensure that any derived cell lines will be amplified only one gene DHFR, namely gene present Desmond-bulleted website. Plasmid "Mandy" was derived from "Molly" by following modifications:

(i) a Synthetic linker was introduced in the middle of the DHFR coding region. This linker introduces a stop codon and shifts the rest of the DHFR coding region outside reading frames, which makes this gene is non-functional.

(ii) part of the HisD gene has been delegated and replaced by PCR-generirovannykh DNA elements in the marker plasmid "Desmond". In Fig.2 shows the location of these DNA elements in the first target plasmid transfer, "Molly". In Fig.3 illustrates a possible layout of the various DNA elements in the genome of the cells of the SSS after the target of the migration and integration of DNA Molly in Desmond-labeled cells SNO. In Fig.9 shows the target plasmid transfer "Mandy".

Designing marker plasmids and plasmids the target of the transfer of the above DNA elements was carried out in accordance with the standard technique of cloning (see, for example, Molecular Cloning, A laboratory manual, J. Sambrook et al., 1987, Cold Spring Harbor Laboratory Press, " Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., 1987, John Wiley & Sons). All plasmids were propagated and maintained in XLI blue E. coli (Stratagene, cat. # 200236). Large-scale plasmid preparations were obtained using the system for DNA purification (Promega Wizard Maxiprep DNA Purification System®) according to manufacturer's instructions.

Example 2

Designing marker Cho cell line

1. Procedures for culturing and transfection of cells produced for marker Cho cell line

Marker plasmid DNA was linearizable by hydrolysis over night at 37With enzyme Bst1107I. The linearized vector precipitated with ethanol and resuspending is isogo hamster cells Cho cells DG44 (Urlaub et al., Som. Cell and Mot. Gen., 12:555-566 (1986) by electroporation as follows.

Cells from exponential growth were harvested by centrifugation, once washed chilled on ice SBC (buffered sucrose solution, 272 mm sucrose, 7 mm sodium phosphate, pH 7.4, 1 mm magnesium chloride), and then resuspendable in SBC to obtain concentrations of 107cells/ml After 15-minute incubation on ice, 0.4 ml of cell suspension was mixed with 40 μg of linearized DNA in a disposable cuvette for electroporation. Cells were subjected to electric shocks by using the manipulator with electrochemically VTH (San Diego, CA), with operating parameters: voltage 230 V, power capacity 400 microfarad and the resistance of 13 Ohms. Then, cells, subjected to electric shocks, was mixed with 20 ml of pre-warmed medium for culturing Cho (Cho-S-SFMII, Gibco/BRL, catalog # 31033-012) and seeded in 96-well plates to the cultivation of tissues. 48 hours after electroporation, the tablets were added to the selective environment (in the case of transfection with plasmid Desmond, the selective medium was medium CHO-S-SFMII without gipoksantina or thymidine, to which was added 2 mm histidinol (Sigma catalog # H6647)). These tablets were maintained in selective medium, uplo is hatov, and, finally, was replicated in roller 120 ml-flasks, where they were in the selective medium.

Example 3

Characterization of labeled cell lines SNO

(a) southern analysis

Genomic DNA was isolated from all steadily growing Desmond-labeled cells SNO. DNA was isolated using a set of Easy Invitrogen® DNA kit according to the manufacturer's instructions. Then, genomic DNA is hydrolyzed by the enzyme HindiI II overnight at 37With, and subjected to southern analysis using PCR-generated labeled digoxigenin probe specific to the DHFR gene. Hybridization and washing was performed using the kit for hybridization, Boehringer Mannhiem''s DIG easy hyb (catalog # 1603558), washing DIG Wash and block buffer Block Buffer Set (catalog # 1585762) according to the manufacturer's instructions. It was assumed that DNA samples containing one strip, for hybridization with DHFR-probe, represented Desmond-clones originating from a single cell, which had a single integrated copy of the plasmid. These clones were retained for subsequent analyses. From all 45 HisD resistant selected cell lines, only 5 were odnokorennye integrante. In Fig.4 shows a southern blot, stereoscopically from all odnoletnih Desmond-clones using TRIzol reagent (Gibco/BRL, cat # 15596026) according to the manufacturer's instructions. 10-20 µg RNA from each clone were analyzed in duplicate formaldehyde gels. The resulting blots were probed using PCR-generated labeled digoxigenin DNA probes for (i) DHFR-transcript, (ii) HisD-transcript, and (iii) CAD transcript. CAD is trifunctionally protein involved in the biosynthesis of uridine (Wahl et al., J. Biol. Chem., 254, 17:8679-8689 (1979)), and the same is expressed in cells of all types. In this case, it was used as an internal control to facilitate a quantitative assessment of the loading of RNA. Hybridization and washing was performed using the above reagents Boehringer Mannhiem. The results of the Northern blot analysis shown in Fig.5. Odnokabinnom Desmond-clone detecting the highest levels of both HisD and DHFR-transcript, was a clone IS shown in track 4 on both panels of the figure. This clone was named "GM cell line and used for subsequent experiments targeted gene transfer in SNO, examples of which are presented in the following chapters.

Example 4

Expression of antibodies against CD20 in Desmond-labeled cells SNO

SW, chimeric antibody, which recognizes the antigen CD20 NOC-fragment 4.1, etc., of O., containing the genes for the light and heavy chains SV, together with the necessary regulatory elements (eukaryotic promoter and polyadenylation signals) embedded in the artificial intron created between exons 1 and 2 of the neo gene, present in the cloning vector, derived from pBR. This newly generated DNA fragment 5, etc., O. (containing neo-exon 1, SV and neo-exon 2) were cut and used to build the target plasmids transfer Molly. Other DNA elements used to construct plasmids Molly, were identical to the items used to construct the marker plasmid Desmond identified above. Full map of plasmid Molly shown in Fig.2.

Vector target transfer Molly was linearized, and then transfected by hydrolysis enzymes kpni restriction sites and PacI, precipitated with ethanol and resuspendable in sterile TE to a concentration of 1.5 mg/ml Linearized plasmid was introduced into exponentially growing Desmond-labeled cells, mainly, as described above, except that in each stage of electroporation used 80 µg DNA. Forty-eight hours after electroporation, in 96-well tablets were added selective environment - CHO-SSFMII, to which was added 400 μg/ml gene is ka in some of the holes did not experience the growth of cells. It is assumed that this increase is the result of clonal expansion of a single S-resistant cells. Supernatant from all G418-resistant wells analyzed for the production of SV using standard ELISA techniques, and all productive clones were eventually reproduced in roller 120 ml-flasks, and then analyzed.

Characterization of antibody-secreting cells-targets

In this experiment, there were just 50 electroporation using the plasmid target transfer Molly, each of which was made into a separate 96-well plates. They received a total of 10 viable clones secreting antibody against CD20, and these clones were propagated in roller 120 ml flasks. From all clones were isolated genomic DNA was carried out by southern analysis in order to determine whether these clones only the events of homologous recombination, or in these same cells occur, in addition, events random integration. Methods DNA extraction and southern hybridization described in the previous section. Genomic DNA hydrolyzed EcoRI and probed with PCR-generated labeled digoxigenin probe specific for a segment of the constant region of the heavy chain CD20. The results of this Simuusa with D20-probe, indicating that in these cells occur only in the event of homologous recombination. Two of the ten clones, 24G2 and S, revealed the presence of additional bands (strips), indicating that in this genome, in addition, there is a random integration.

For all ten clones were evaluated expression levels of antibodies against CD20, and data from these assessments are presented in table 1.

Levels of expression, secreted by individual clones, expressed in pilgrammage per cell per day (PG/CL/day) and represent the levels obtained from three ELISA-tests performed on samples taken from the roller 120 ml flasks.

As can be seen from these data, between the clones with the highest expression and clones with the lowest expression, secretion of antibodies varies approximately ten times. This result was somewhat surprising as we expected to obtain similar levels of expression for all clones, based on the fact that all anti-D20-genes were integrated in the same Desmond-marked the site. However, this observed range of expression is extremely small compared with that observed using any traditional IU 20F4, producing the highest level adenocarinoma of integrant, was selected for further studies. Table 2 presents the results of the ELISA and the data on the cultivation of cells derived from seven stages of production of this clone.

Clone 20F4 were seeded at a concentration of 210 ml roller 120 ml-flask on day 0. In the next six days, count the cells, while the counting was performed in duplicate and from this flask was taking 1 ml samples of the supernatant and analyzed using ELISA for secreted antibodies against CD20.

Based on the ELISA data, the clone secreterial on average 3-5 PG antibodies per cell per day. This level was similar to the level obtained from other vysokoagressivnyh odnoletnih clones obtained in our laboratory using a previously constructed translational weakened vectors for random integration. This result suggests that:

(1) site present in the genome of cells SNO marked Desmond-marking vector is highly transcriptionally active, and therefore it is the most suitable site from which expressed resettle using the vectors of the present invention and its frequency is, high enough to make this system feasible and desirable alternative methods of random integration.

To further illustrate the effectiveness of this system, we have also demonstrated that this site may be amplified, which may even lead to higher levels of gene expression and protein secretion. Amplification was carried out by seeding G cells in serial dilutions, starting with a density of 2.5104cells/ml in 96-well plates to the cultivation of tissues, and culturing these cells in media (CHO-SSFMII), to which was added 5, 10, 15 or 20 nm methotrexate. Antitelomerase clones were skanirovali using standard ELISA techniques, and most vysokoplodorodnye clones were multiplied, and then analyzed. The results of this experiment for amplification are presented in table 3.

Amplification using methotrexate 20F4 were performed as described in the text, using concentrations of methotrexate listed in the table. 3 Supernatant from all surviving 96-hole colonies were analyzed using ELISA, and the range of expression levels of antibodies against CD20, has ProductModel in roller 120 ml flasks and supernatant from spin-cultures were subjected to several tests ELISA for determination of expression levels (PG/cell/day), shown in column 5.

The data obtained clearly indicate that this site may be amplified in the presence of methotrexate. It was found that the clones from the 10 and 15 nm amplification produce levels of 15-20 PG/cell/day.

Clone 15 nm, indicated 20F4-15A5, was selected as the most vysokoaktivnye cell lines. This clone was obtained from a 96-hole of the tablet, in which the growth was observed only in 22 holes, and therefore, we can assume that he comes from a single cell. Clone 15 nm, indicated 20F4-15A5, was selected as the most vysokoaktivnye cell lines. This clone was obtained from a 96-hole of the tablet, in which the growth was observed only in 22 holes, and therefore, we can assume that he comes from a single cell. Then the clone was subjected to additional cycles of amplification using methotrexate. As described above, the culture in serial dilutions were sown in 96-well plates, and cultured in the medium of CHO-SS-FMII, to which was added 200, 300, or 400 nm methotrexate. Surviving clones were skanirovaniya using ELISA, and several wysokoprocentowych clones were propagated to obtain the spin-cultures, and then analyzed. The results of this second experiment of anorexia 20F4-15A5 conducted, as described in the text, using concentrations of methotrexate listed in the table. 4 Clones in the wells with the highest degree of production numbers listed in column 4, multiplied in roller 120 ml flasks. The levels of expression from cell lines derived from these holes, expressed in PG/cell / day and given in column 5.

The highest producing clones were obtained by amplification using 250 nm methotrexate. 250 nm-Clone, 20F4-15A5-250A6, was obtained from 96-well to the tablet, in which growth was detected only in the cells, and therefore, we can assume that it originates from a single cell. The data of tables 3 and 4, taken together, clearly show that two cycles of amplification using methotrexate enough to achieve the levels of expression of 60 PG/KL/day, which is approaching the maximum level of secretion of immunoglobulin in mammalian cells (Reff, M. E., Curr. Opin. Biotech., 4:573-576 (1993)). The ability to achieve this level of expression only two stages of amplification increases the value of this system of homologous recombination. Typically, in the methods of random integration to achieve such a level of expression requires more than two stages of amplification, and he shall then give a more effective and requires less time way of achieving a high level of gene expression in mammalian cells.

Example 5

The expression of CD23 antibodies against human rights in Desmond-labeled cells SNO

CD23 is a low-affinity IgE receptor, which mediates the binding of IgE from b - and T-lymphocytes (Sutton, B. J. & Gould, H. J., Nature, 366:421-428 (1993)). Monoclonal antibody against human CD23 E is a monoclonal antibody gamma-1, which was recently cloned and expressed in our laboratory. This antibody is described in co assigned application reg. No. 08/803085, filed February 20, 1997.

The genes of the heavy and light chain antibodies E were cloned in expressing vector mammal N5KG1 obtained from the vector NEOSPLA (Barnett et al., Antibody Expression and Endineering, H. Y. Yang & T. Imanaka, eds., p. 27-40 (1995)), and then these genes were introduced two modifications. Recently we observed a slightly higher secretion light chain immunoglobulin compared with heavy chains in other expressing the structures obtained in the laboratory (Reff et al., unpublished observations). In an attempt to compensate for this deficit, we modified heavy chain gene E by adding more strong promoter/enhancer directly above the site of initiation. In subsequent stages, 2,9, etc., O. - DNA fragment containing modified genes Lamego vector Molly and electroporation in cells SNO, containing Desmond 15C9, carried out mainly as described in the previous section.

In accordance with the previously described scheme, one modification was made in the culture medium of the type. Desmond-labeled cells SNO were cultured in the medium of CD-CHO, containing no protein (Gibco-BRL, catalog # AS21206), to which was added 3 mg/l recombinant human insulin (3 mg/ml stock solution, Gibco-BRL, catalog # AS22057), and 8 mm L-glutamine (200 mg/ml stock solution, Gibco-BRL, catalog # 25030-81). Then transfected cells were selected in the above environment, to which was added 400 μg/ml of geneticin. This experiment was carried out 20 electroporate, and the obtained cells were seeded in 96-well plates to the cultivation of tissues. Cells were grown and secretively antibody against CD23 only 68 holes, all of which, presumably, was represented by clones derived from one S cells. Twelve of these clones were propagated in roller 120 ml flasks for subsequent analysis. We assume that the increased number of clones selected in this experiment (68 compared with 10 for antibodies against CD20, described in Example 4), due to higher efficiency of cloning and survival of cells cultured in media is skilled in the spin-culture, was within 0.5 to 3 PG/CL/day, which almost corresponds to the levels observed for clones secreting antibody against CD20. To increase levels of expression of the clone producing the highest levels of antibodies against CD23 and marked N, he was subjected to amplification using methotrexate. This amplification was carried out in a manner analogous to the method used for anti-CD20-Clonie described in Example 4. Exponentially growing N cells in serial dilutions were seeded in 96-well plates to the cultivation of tissues and cultured in the medium of CD-CHO, to which was added 3 mg/l insulin, 8 mm glutamine and 30, 35 or 40 nm methotrexate. The results of this experiment for amplification are presented in table 5.

Most vysokoagressivnyh obtained clone was 30 nm clone isolated from a tablet, in which growth was found 22 wells. This clone, designated 4H12-30G5, replicable secretarial 18-22 PG antibodies per cell per day. This same interval of expression levels was observed for the first amplification anti-S clone 20F4 (clone 20F4-15AS, which was produced 15-18 PG/CL/day, as described in Example 4). These data are used for additional pod and effective. Second, the amplification of this 30 nm-cell lines is underway. It is expected that the saturation levels of expression can be achieved for antibodies against CD23 only two stages of amplification, as in the case of antibodies against CD20.

Example 6

Expression immunoadhesin in Desmond-labeled cells SNO

CTLA-4, a member of the superfamily, 1D, found on the surface of T-lymphocytes, and, obviously, plays a role in antigen-specific activation of T cells (Dariavach et al., Eur. J. Iinmunol., 18:1901-1905 (1988); and Linsley et al., J. Exp. Med., 174:561-569 (1991)). To further study the specific role of the molecule CTLA-4 pathway activation, was designed soluble protein containing the extracellular domain of CTLA-4, attached to a truncated form of the constant region of human IgGI (Linsley et al., ibid.). Recently we carried out expression of this fused protein, CTLA-4-Ig in expressing vector BLECH1 mammal-derived plasmids NEOSPA (Barnett et al., in Antibody Expression and Engineering, H. Y. Yang & T. Imanaka, eds., p. 27-40 (1995)). 800 p. O. fragment encoding CTLA-4-Ig, was isolated from this vector, and inserted between the SacII and BgIII sites in Molly.

Getting CTLA-4-Ig-Molly and electroporation in cells SNO Desmond-clone C was carried out as described in the previous example relating to anthralin tablets, as described previously. From 96-well plates were selected eighteen CTLA-4-expressing holes and placed directly in the roller 120 ml-flask. To determine how many homologous clones contain additional random integrant, was conducted by southern analysis of genomic DNA isolated from these clones. Genomic DNA is hydrolyzed by the enzyme BglII and probed with PCR-generated and labeled digoxigenin probe for a constant region of human IgGI. The results of this analysis indicated that 85% of CTLA-4-clones was only homologous integrante; the remaining 15% contained one additional random integrant. This result confirms the facts discovered as a result of expression of anti-D20-clone and discussed above, where 80% of the clones were only homologous integrante. Therefore, from this we can conclude that this expression system allows replicable get only targeted homologous integrate at least 80% of all produced clones.

The levels of expression for homologous CTLA-4-Ig-clones were in the range of 8-12 PG/CL/day. This range is slightly higher than the range specified for clones of antibodies against CD20 and CD23 antibodies against, discuss the th vector system also provides significantly higher levels of expression, than those that were obtained for immunoglobulins. Currently, we cannot explain this observation.

Example 7

Directed the introduction of antibodies against CD20 in another Desmond-labeled cell line Cho

As described in the previous section, received 5 odnoletnih Desmond-labeled cell lines Cho (see Fig.4 and 5). To illustrate that the success of our strategy is the target of transfer is not due to some unique property Desmond-clone S and is not limited to this clone, we introduced anti-D20-plasmid Molly in Desmond-clone W (lane 6 in Fig.4, lane 1 in Fig.5). Obtaining DNA Molly and electroporation in Desmond-clone B was carried out exactly as described in the previous example related to the antibody against CD20. In this experiment we got one homologous integrant. This clone was propagated in roller 120-ml flask, where it was produced in an average of 1.2 PG/KL/day of antibodies against CD20. This level of expression is significantly smaller than the one we observed with the use of plasmids Molly entered by the target transfer Desmond-clone S. However, this result might have been expected, considering we conducted Northern analysis Desmond-clones. As can be seen from Fig.5, the levels of mRNA from clone W about active, as in the clone C. Therefore, this experiment not only demonstrates the reproducibility of this system - presumably, any bulleted website Desmond can be entered by the target transfer using Molly, but also confirms the data, Northern blot analysis, indicating that the site in Desmond-clone C is the most transcriptionally active.

Based on the above it should be noted that although specific variants of the present invention have been described herein for purposes of illustration, however, in the present invention can be made of various modifications without leaving the scope of the invention. In accordance with this present invention is not limited to enclosed items by the claims.

Claims

1. The method of embedding the desired DNA into the genome of mammalian cells in vitro, comprising the following stages: (i) transfection or transformation of mammalian cells, in tissue culture, the first plasmid (“marker plasmid) containing following sequence: (a) a region of DNA that is heterologous to the genome of mammalian cells and which when integrated into the genome of mammalian cells provides a unique site for Gamaleya least other selective marker DNA, providing selection of mammalian cells that have been successfully integrated marker plasmid; (ii) selection of cells that contain the marker plasmid integrated into the genome; (iii) the transfection or transformation of these selected mammalian cells the second plasmid (target plasmid) containing following sequence: (a) a region of DNA that is identical or highly homologous to a unique region in the marker plasmid so that this region of DNA can recombine with the indicated DNA by homologous recombination; (b) a DNA fragment, encoding the second portion of the first selective marker protein marker plasmids, where the first selective marker protein is produced only when the specified second part is expressed together with the specified first part of the first selective marker protein; (c) DNA that needs to be incorporated into the genome of mammalian cells; and (iv) the selection of cells that contain the target plasmid integrated into the target site by screening for the expression of the first selective marker protein.

2. The method according to p. 1, where a DNA fragment coding for the fragment of the first selective marker exon is the CSO of the first selective marker.

4. The method according to p. 3, where at least one DNA encoding a desired protein is embedded between these exons specified first selective marker contained in the target plasmid.

5. The method according to p. 4, where the target plasmid further comprises DNA encoding a dominant selective marker, built between exons specified first selective marker contained in the target plasmid, to ensure co-amplification of DNA that encodes a desired protein.

6. The method according to p. 3, where the first dominant selective marker selected from the group consisting of neomycin-phosphotransferase, histidinol-dehydrogenase, dihydrofolate-reductase, hygromycin-phosphotransferase, timedancing of herpes simplex virus, adenozindezaminazy, glutamine synthase and gipoksantin-guaninephosphoribosyltransferase.

7. The method according to p. 4, where the desired protein is a protein of a mammal.

8. The method according to p. 7, where the specified protein is an immunoglobulin.

9. The method according to p. 1, further comprising determining levels of RNA selective marker encoded by the DNA of (C) marker plasmids, before integration of the target vector.

10. The method according to p. 9, where other selective marker contained in the marker plasmid is domin herpes simplex, hygromycin-phosphotransferase, adenozindezaminazy and glutamine synthase.

11. The method according to p. 1, where the cell of a mammal selected from the group consisting of cells of the Chinese hamster ovary (Cho), myeloma cells, kidney cells baby hamster, COS cells, NSO cells, HeLa cells and cells NIH ZTZ.

12. The method according to p. 11, where the specified cell is a cell SNO.

13. The method according to p. 1, where the marker plasmid contains the third exon of the gene neomycin-phosphotransferase and the target plasmid containing the first two exons of the gene neomycin-phosphotransferase.

14. The method according to p. 1, where the marker plasmid also contains rare restriction endonuclease sequence embedded within the region of homology.

15. The method according to p. 1, where the unique region of DNA that provides the homologous recombination is bacterial DNA, viral DNA or synthetic DNA.

16. The method according to p. 1, where the unique region of DNA that provides the homologous recombination is at least 300 nucleotides.

17. The method according to p. 16, where the unique region of DNA has a size in the range from about 300 nucleotides to 20 thousand base pairs.

18. The method according to p. 17, where the unique region of DNA has a size of, preferably, in the range from 2 to 10 thousand pairs

20. The method according to p. 1, where the unique region of DNA that provides the homologous recombination is bacterial DNA, DNA insect virus DNA or synthetic DNA.

21. The method according to p. 20, where the unique region of DNA contains no functional genes.

22. Vector system for embedding the desired DNA into the genome of mammalian cells in tissue culture, comprising (i) a first plasmid (“marker plasmid) containing at least the following sequence: (a) a region of DNA that is heterologous to the genome of mammalian cells, and which, when integrated into the genome of mammalian cells provides a unique site for homologous recombination; (b) a DNA fragment encoding the first portion of the first selective marker protein; and (c) at least one other selective marker DNA providing for selection of mammalian cells that have been successfully integrated marker plasmid; (ii) a second plasmid (target plasmid) containing at least the following sequence: (a) a region of DNA that is identical or highly homologous to a unique region in the marker plasmid so that this region of DNA can recombine with the indicated DNA group is protein marker plasmids, where the first selective marker protein is produced only when the specified second part is expressed together with the specified first part of the first selective marker protein; and (c) DNA that needs to be incorporated into the genome of mammalian cells.

23. The vector system according to p. 22, where DNA fragment, encoding a portion of the first selective marker is the exon dominant selective marker.

24. Vector system for p. 23, where the second plasmid contains the remaining exons of the specified first selective marker.

25. The vector system according to p. 24, where at least one DNA encoding a desired protein is embedded between these exons specified first selective marker contained in the target plasmid.

26. The vector system according to p. 24, where the target plasmid further comprises DNA encoding a dominant selective marker, built between exons specified first selective marker contained in the target plasmid, to ensure co-amplification of DNA that encodes a desired protein.

27. The vector system according to p. 24, where the first dominant selective marker selected from the group consisting of neomycin-phosphotransferase, histidinol-dehydrogenase, dihydrofolate-reductase and gipoksantin-guaninephosphoribosyltransferase.

28. Vector system for p. 25, where the desired protein is a protein of a mammal.

29. Vector system for p. 28, where the specified protein is an immunoglobulin.

30. The vector system according to p. 22, where other selective marker contained in the marker plasmid, is a dominant selective marker selected from the group consisting of histidinol-dehydrogenase, timedancing of herpes simplex virus, hygromycin-phosphotransferase, adenozindezaminazy and glutamine synthase.

31. The vector system according to p. 22, which integrates the desired DNA target site in the genome of mammalian cells selected from the group consisting of cells of the Chinese hamster ovary (Cho), myeloma cells, kidney cells baby hamster, COS cells, NSO cells, HeLa cells and cells NIH ZTZ.

32. Vector system for p. 31, where the specified cell of a mammal cell is SNO.

33. The vector system according to p. 22, where the marker plasmid contains the third exon of the gene neomycin-phosphotransferase and the target plasmid contains the first two exons of the gene neomycin-phosphotransferase.

34. The vector system according to p. 22, where the marker plasmid further comprises a rare restriction endonuclease sequence embedded in abominatio, is bacterial DNA, viral DNA or synthetic DNA.

36. The vector system according to p. 22, where the unique region of DNA (a) contained in the marker plasmid vector system is at least 300 nucleotides.

37. The vector system according to p. 36, where the unique region of DNA has a size in the range from about 300 nucleotides to 20 thousand base pairs.

38. Vector system for p. 37, where the unique region of DNA, preferably has a size in the range from 2 to 10 thousand base pairs.

39. The vector system according to p. 22, where the first selective marker DNA separated by at least three exons.

40. The vector system according to p. 22, which is a unique region of DNA that provides the homologous recombination is bacterial DNA, DNA insect virus DNA or synthetic DNA.

41. The vector system according to p. 40, where the unique region of DNA does not contain functional genes.

Priority items:

14.03.1997 on PP.1-41.

 

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