MXPA99008363A - Method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same - Google Patents

Abstract

A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Vectors and vector combinations for use in the subject cloning method are also provided.

Description

METHOD FOR INTEGRATING GENES IN SPECIFIC SITES IN - * • 'MAM FERO CELLS BY RECOMBINATION HOMOLOGA Y VECTORS TO PERFORM THE SAME Field of the Invention The present invention relates to a targeting process for the integration of a desired exogenous DNA to a specific location within the genome of a mammalian cell. More specifically, the invention describes a novel method for identifying a target site transcriptionally active ("hot spot") in the genome of the mammal, and inserting a desired DNA at this site by means of homologous recombination. The invention also optionally provides the ability for the amplification of the desired DNA gene at this site by the co-integration of an amplifiable selectable marker, eg, DHFR, in combination with the exogenous DNA. The invention further describes the construction of novel vectors suitable for carrying out the foregoing, and further provides lines of mammalian cells produced by such methods, which contain a desired exogenous DNA integrated into a target hot spot. Ref.031290 Background of the Invention The technology to express recombinant proteins in both prokaryotic and eukaryotic organisms is well established. Mammalian cells offer significant advantages over bacteria or yeasts for the production of proteins, which result from their ability to assemble, glycosylate and correctly posttranslationally modify recombinantly expressed proteins. After transfection in the host cells, the recombinant expression constructs can be maintained as extrachromosomal elements, or they can be integrated into the genome of the host cell. The generation of stably transfected mammalian cell lines usually involves last; a DNA construct that encodes a gene of interest in the company of the gene that provides resistance to the drug (dominant selectable marker), is introduced into the host cell, and the subsequent growth in the presence of the drug allows the selection of the cells that have integrated successfully exogenous DNA. In many cases, the gene of interest is linked to a drug-resistant selectable marker which can ultimately be subjected to an amplification of the gene. The gene that codes for dihydrgfolate reductase - (DHFR) is used most completely for this purpose. The growth of the cells in the presence of methotrexate, a competitive inhibitor of DHFR, leads to an increased DHFR production by means of the amplification of the DHFR gene. As the flanking regions of the DNA will also become amplified, the resulting co-amplification "of a gene linked to DHFR in the transfected cell line can lead to increased protein production, thereby leading to high level expression of the Although this approach has proven to be successful, there are several problems with the system because of the random nature of the "integration event. These problems exist because levels of expression are largely influenced by the effects of the local genetic environment on the gene site, a phenomenon well documented in the literature and generally referred to as "position effects" (for example, see Al-Shawi et al, Mol Cell. Biol. 10: 1192-1198 (1990), Yoshimura et al, Mol Cell Biol., 7: 1296-1299 (1987)). Since the vast majority of mammalian DNA is in a transcriptionally inactive state, random integration methods offer no control over the transcriptional fate of "integrated" DNA.As a consequence, wide variations in the expression level of integrated genes can occur. Depending on the integration site, for example, the integration of exogenous DNA into inactive regions, or transcriptionally "quiet" of the genome, will lead to a small expression or no expression.The integration of the contrast in a transcriptionally active site can lead to a high expression, therefore, when the goal of the work is to obtain a high level of gene expression, as is typically the desired result of genetic engineering methods, it is generally necessary to select large numbers of transfectants to find such a high production clone. Additionally, random integration --- of exogenous DNA in the genome can in some cases alter important cellular genes, leading to an altered phenotype. These factors can make the generation of mammalian cell lines stable of high expression, a complicated and laborious process. Recently, the present applicant's laboratory has described the use of DNA vectors containing dominant selectable markers altered translationally in the expression of the mammalian gene. (This is described in U.S. Serial No. 08 / 147,696 filed on November 3, 1993, recently allowed). These vectors contain an "icine (neo) -phosphotransferase gene translationally altered as the selectable marker, artificially designed to contain an intron within which a DHFR gene in the company of a foreign gene or genes is inserted. These vectors as expression constructs have been found to significantly reduce the total number of colonies resistant to the drugs produced, thus facilitating the selection procedure in relation to conventional mammalian expression vectors. clones obtained using this system are the "high expression" clones. These ^ results are apparently attributable to modifications made to the neo-selectable marker. ~ Due to the translational alteration of the neo gene, the transfected cells will not produce enough neo-protein to survive drug selection, thus reducing the total number of drug-resistant colonies. Additionally, a higher percentage of the clones that survived will contain the expression vector integrated into the sites in the genome where the levels of basal transcription are high, leading to the overproduction of neo, by which the cells are allowed to surpass the alteration of the neo gene. Concomitantly, the genes of interest linked to neo will be subjected to similar high levels of transcription. This same advantage is also true as a result of the artificial intron created within neo; Survival depends on the synthesis of a neo-functional gene, which in turn depends on the correct and efficient union of neo-introns. In addition, these criteria are more likely to be satisfied if the vector DNA has been integrated into a region which is already highly transcriptionally active. Following integration of the vector into a transcriptionally active region, the amplification of the gene is effected by selection for the DHFR gene. Using this system, it has been possible to obtain selected clones using low levels of methotrexate (50 nM), which contains few copies (< 10) of the vector which will secrete high levels of protein (> 55pg / cell / day). In addition, this can be achieved in a relatively short period of time. However, the success in amplification is variable. Some transcriptionally active sites can not be amplified and therefore the frequency and extent of amplification of a particular site is not predictable. In summary, the use of these transcriptionally altered vectors represents a significant improvement over other methods of random integration. However, as described, the problem of lack of control over the integration site remains a matter of significant interest. One approach to overcoming the problems of random integration is by targeting the gene, whereby the exogenous DNA is directed to a specific site within the host genome. The exogenous DNA is inserted through the homologous recombination that occurs between the DNA sequences in the expression vector and the corresponding homologous sequence in the genome. However, although this type of recombination occurs at a high frequency naturally in yeast and other fungal organisms, in eukaryotic organisms it is an extremely rare event. In mammalian cells, the frequency of homologous versus non-homologous recombination (random integration) is reported at a range of 1/100 to 1/5000 (for example, see Capecchi, Science, 244: 1288-1292 (1989)). ); Morro and Kucherlapati, Curr. Op. Biotech., 4: 577-582 (1993)).

One of the closest reports describing homologous recombination in mammalian cells comprised an artificial system created in mouse fibroblasts (Thomas et al., Cell, 44: 419-428 (1986)). A cell line containing a non-functional, mutated version of the neo gene integrated into the host genome was created, and subsequently it was targeted as a second non-functional copy of neo containing a different mutation. The reconstruction of a neo-functional gene occurs only because of the location as a target of the gene. The homologous recombinants were identified "selecting G418 resistant cells, and confirmed by analysis of genomic DNA isolated from resistant clones. * Recently, the use of homologous recombination to replace the heavy and light immunoglobulin genes in the endogenous sites in the cells that secrete antibodies, has has been reported (US Patent No. 5,202,238, Fell et al., (1993).) However, this particular approach is not widely applicable, because it is limited to the production of immunoglobulins in cells which endogenously express immunoglobulins, eg B cells and myeloma cells Also, expression is limited to single-copy gene levels, because co-amplification after homologous recombination is not included The method is further complicated by the fact that two separate integration events are required to produce a functional immunoglobulin: one for the liger chain gene a followed by one for the heavy chain gene. A further example of this type of system has been reported in NS / O cells, where recombinant immunoglobulins are expressed by homologous recombination at the gamma 2A site of immunoglobulin (Hollis _et al, International patent application # PCT / IB95 ( 00014) .The levels of ^ expression obtained from this site were extremely high - of the order of 20 pg / cell / day from a single-partintegrant. However, as in the previous example, expression is limited to this level because an amplifiable gene is not cointegrated in this system. Also, other researchers have reported an aberrant glycosylation of recombinant proteins expressed in NS / O cells (for example, see Flesher et al, Biotech and Bioeng., 48: 399-407 (1995)), which limits the applicability of this approach. The cre-loxP recombination system of bacteriophage Pl has recently been adapted and used as a means of targeting a target in eukaryotic cells. Specifically, the specific integration of the exogenous DNA site in the Chinese hamster ovary (CHO) cell genome using the ere recombinase and a series of lox-containing vectors has been described. (Fukushige and Sauer, Proc. Nati, Acad. Sci. USA, 89: 7905-7909 (1992)). This system is attractive because it provides reproducible expression in the same chromosomal location. However, no effort has been made to identify a chromosomal site from which the expression of the gene is optimal, and as in the previous example, the expression is limited to unique copy levels in this system. Also, this is complicated by the fact that it is necessary to provide the expression of a functional recombinase enzyme in the mammalian cell. The use of homologous recombination between an introduced DNA sequence and its endogenous chromosomal site has also been reported to provide a useful means of genetic manipulation in mammalian cells, as well as in yeast cells. (See for example, Bradley et al, Meth. Enzimol., 223: 855-879 (1993); Capecchi, Science, 244: 1288-1292 (1989); Rothstein et al, Meth. Enzymol., 194: 281-301 (1991)). To date, most studies of the location of a target of the mammalian gene have been directed towards the break ("out of combat") of the gene or site-specific mutagenesis of the selected target gene in the cells of the mouse embryonic stem (ES). The creation of these mouse models "out of combat" has made it possible for scientists to examine the issues of structural function and examine the biological importance of a large number of mouse genes. This field of research has important implications in terms of potential applications of gene therapy. As well, vectors have recently been reported by Celltech (Kent, U.K.) which are purposely • targeted or targeted to transcriptionally activate the active sites in the cells of the cell.

NSO, which do not require amplification of the gene (Peakman et al, Hum.Antibod.Hybridomas, 5: 65-74 J1994)). However, the levels of immunoglobulin secretion in these unamplified cells have not been reported to exceed 20 pg / cell / day, whereas in the amplified CHO cells, levels as high as 100 pg / cell / day can be obtained. (Id.). It could be highly desirable to develop a system for locating a target of the gene which reproducibly provides for the integration of exogenous DNA at a predetermined site in the genome that is known to be transcriptionally active. Also, it could be desirable if such a targeting system of the gene could additionally facilitate co-amplification of the inserted DNA after integration. The design of such a system could allow the high level and reproducible expression of any cloned gene of interest in a mammalian cell, and undoubtedly would be of significant interest to many researchers. In this application, a novel mammalian expression system is provided, based on homologous recombination occurring between two artificial substrates contained in two different vectors. Specifically, this system uses a combination of two novel mammalian expression vectors, referred to as a "tag" vector and a "target location" vector. Essentially, the tag vector makes it possible to identify and label a site in the mammalian genome which is transcriptionally active, i.e., a site in which the expression levels of the gene are high. This site can be considered a "hot spot" in the genome. After integration of the vector tag, the object expression system makes it possible for another DNA to be integrated into this site, ie, the target or target location vector, by means of the homologous recombination that occurs between the sequences of DNA common to both vectors. This system produces significant advantages over other homologous matching systems. Unlike most homologous systems used in mammalian cells, this system does not exhibit a background. "Therefore, cells that have only undergone random integration of the vector do not survive selection. any gene of interest cloned in the target location plasmid is expressed at high levels from the labeled hot spot.As a result, the method object of gene expression substantially or completely eliminates the inherent problems of random integration, described in detail In addition, this system provides reproducible and high-level expression of any recombinant protein in the same site transcriptionally active in the mammalian genome.In addition, the amplification of the gene can be effected in this particular transcriptionally active site including a selectable marker. dominant, amplifiable (for example DHFR) as part of the marc vector ation.

Objects of the Invention By. Consequently, it is an object of the invention to provide an improved method for targeting a desired DNA to a specific site in a mammalian cell. It is a more specific object of the invention to provide a novel method for targeting a desired DNA with respect to a specific site in a mammalian cell by means of homologous recombination. It is another specific object of the invention to provide novel vectors to achieve site specific integration of a desired DNA in a mammalian cell.It is yet another object of the invention to provide novel mammalian cell lines which contain a desired DNA. integrated into a predetermined site which provides high expression.It is a more specific object of the invention to provide a novel method to achieve site-specific integration of a desired DNA in a Chinese hamster ovary (CHO) cell. another more specific object of the invention to provide a novel method for integrating the immunoglobulin genes, or any other genes, in mammalian cells at predetermined chromosomal sites that provide high expression. It is another specific object of the invention to provide novel vectors and combinations of vectors suitable for the integration of immunoglobulin genes into mammalian cells at predetermined sites that provide high expression. It is another object of the invention to provide mammalian cell lines containing integrated immunoglobulin genes at predetermined sites that provide high expression. It is a still more specific object of the invention to provide a novel method for integrating immunoglobulin genes that provide high expression, as well as novel vectors and vector combinations that provide such integration of immunoglobulin genes into CHO cells. Furthermore, it is a specific object of the invention to provide lines of novel CHO cells which contain integrated immunoglobulin genes at predetermined sites that provide high expression, and which have been amplified by selection of methotrexate to secrete even larger amounts of immunoglobulins. functional Brief Description of the Figures Figure 1 shows a map of a labeling plasmid according to the invention, referred to as Desmond. The plasmid is shown in the circular form (la) as well as a "linearized" version used for transfection (lb.) Figure 2 (a) shows a map of a target location plasmid referred to as "Molly." Molly is shown here coding for the anti-immunoglobulin genes. CD20, the expression of "which is described in Example 1. Figure 2 (b) shows a linearized version of Molly, after digestion with the restriction enzymes Kpnl and Pací. This linearized form was used for transfection. Figure 3 shows the potential alignment between the Desmond sequences integrated into the CHO genome, and the Molly sequence of the target entry location. A potential Molly array integrated in Desmond after homologous recombination is also presented. Figure 4 shows a Southern analysis of the single-copy Desmond clones. The samples are as follows: Strip 1: DNA size marker? HindIII Strip 2: Desmond Faja 3 clone 10F3 3: Desmond Clone 10C12 clone 4: Desmond Clone 15C9 clone 5: Desmond Clone 14B5 clone 6: Desmond clone 9B2 Figure 5 shows a Northern analysis of the single-copy Desmond clones. The samples are as follows: Panel A: northern probed with CAD and DHFR probes, as indicated on the Figure. Panel B: northern duplicate, probed with CAD and HisD probes, as indicated. The samples of RNA loaded in panels A and B are as follows: Strip 1: clone 9B2, strip 2; clone 10C12, strip 3; clone 14B5, strip 4; clone 15C9, strip 5; Control RNA from CHO transfected with a plasmid containing HisD and DHFR, strip 6; CHO not transfected. Figure 6 shows a Southern analysis of the clones resulting from the homologous integration of Molly in Desmond. The samples are as follows: Strip 1: DNA size markers of? HindIII, Strip 2: 20F4, strip 3; 5F9, band 4; 21C7, band 5; 24G2, strip 6; 25E1, band 7, 28C9, band 8; 29F9, strip 9; 39G11, strip 10; 42F9, band 11, 50G10, band 12, DNA of the Molly plasmid, linearized with BglII (upper band) and cut with BglII and KpnI (lower band), band 13; Desmond not transfected. Figures 7A to 7G contain the Sequence Listing for Desmond. Figures 8A to 81 contain the Sequence Listing for anti-CD20 containing Molly. Figure 9 contains a map of the target location plasmid, "Mandy", shown here encoding the anti-CD23 genes, the expression of which is described in Example 5. Figures 10A_a ION contain the sequence listing of "Mandy" "containing the anti-CD23 genes as described in Example 5.

Detailed description of the invention The invention provides a novel method for integrating a desired exogenous DNA into a target site within the genome of a mammalian cell by means of homologous recombination. Also, the invention "provides novel vectors to achieve site-specific integration of a DNA at a target site in the genome of a mammalian cell." More specifically, the object cloning method provides site-specific integration. of a desired DNA in a mammalian cell by transfection of such a cell with a "marker plasmid" which contains a unique sequence that is foreign to the mammalian cell genome and which provides a substrate for homologous recombination, followed by transfection with a "target or target plasmid" which contains a sequence which provides homologous recombination with the unique sequence contained in the marker plasmid, and which further comprises a desired DNA to be integrated into the cell of the mammal Typically, the integrated DNA will encode a protein of interest, such as an immunoglobulin or other mammalian glycoprotein. Fero _secretada. The system of homologous recombination "exemplified uses the neomycin phosphotransferase gene as a dominant selectable marker. This particular marker was used based on the following observations previously published; (i) the demonstrated ability to target and restore function to a mutated version of the neo gene (cited at the beginning) and (ii) the development of the invention of translationally altered expression vectors, in which the neo gene has been artificially created as two exons with a gene of interest inserted into the intervening intron; the exons of neo are transladados and united correctly in vivo, producing a functional protein and for which they confer the resistance of G418 on the resulting cellular population. In this application, the neo gene is divided into three exons. The third neo-exon is present on the "marker" plasmid and becomes -integrated into the genome of the host cell during the integration of the marker plasmid into the mammalian cells. Exons 1 and 2 are present in the targeting plasmid as target or target, and are separated by an intervening intron within which at least one gene of interest is cloned. The homologous recombination of the vector of interest with the integrated labeling vector results in or leads to the correct union of all three exons of the neo gene and thus the expression of a neo protein. 'functional (as determined by selection for colonies resistant to G418). Previous to the design of "Common expression system, we have experimentally tested the functionality of such a triple-linked construct in mammalian cells." The results of this control experiment indicated that the three neo exons were "properly bound and therefore suggested the feasibility of the invention object.

However, although the present invention is exemplified using the neo gene, and more specifically a triple-linked neo gene, the general methodology must be effective with other dominant selectable markers. As described in more detail infra, the present invention produces numerous advantages with respect to the methods of expression of conventional genes, including methods of both location and target of the gene as well as random integration. Specifically, the subject invention provides a method which reproducibly allows site-specific integration of a desired DNA in a transcriptionally active domain of a mammalian cell. In addition, because the object method introduces an artificial region of "homology" which acts as a single substrate for homologous recombination and the insertion of a desired DNA, the efficacy of the subject invention does not require the cell to contain or express endogenously ~ a specific DNA. Accordingly, the method is applicable generically to all mammalian cells, and can be used to express any type of recombinant protein. The use of a triply-linked selectable marker, for example, the triple-linked triple-exemplified construct, ensures that all G418-resistant colonies produced will arise from a homologous recombination event (the random members will not produce a neo-functional gene and consequently not will survive the selection of G418). Accordingly, the subject invention makes it easy to select the desired homologous event. In addition, the frequency of additional random integrations in a cell that has undergone a homologous recombination event appears to be low. Based on the foregoing, it is evident that a significant advantage of the invention is that it substantially reduces the number of colonies that need to be selected to identify high producing clones, i.e., cell lines containing a desired DNA which secretes the protein corresponding to high levels. On average, clones containing the desired integrated DNA can be identified by selecting approximately 5 to 20 colonies (compared to several thousand that must be selected when using standard random integration techniques, or several hundred using the intronic insertion vectors previously described ). Additionally, when the site of integration is preselected and comprises a transcriptionally active domain, all exogenous DNA expressed at this site must produce high, ie comparable, levels of the protein of interest. In addition, the subject invention is further advantageous in that it makes it possible for an amplifiable gene to be inserted in the integration of the marker vector. Accordingly, when a desired gene is targeted for this site by means of homologous recombination, the subject invention allows the expression of the gene to be further enhanced by the amplification of the gene. In this regard, it has been reported from the literature that different genomic sites have different capacities for gene amplification (Meinkoth et al, Mol Cell Biol., 7: 1415-1424 (1987)). Therefore, this technique is advantageous additionally because it allows the placement of a desired gene of interest at a specific site that is both transcriptionally active and easily amplified. Therefore, this should significantly reduce the amount of time required to isolate such high producers. Specifically, although conventional methods for the construction of high expression mammalian cell lines can take 6 to 9 months, the present invention allows such clones to be isolated on average ---- after only about 3-6 months. This is due to the fact that conventionally isolated clones must undergo at least three rounds of amplification of the drug resistant gene to achieve satisfactory levels of gene expression. When the homologously produced clones are generated from a preselected site which is a high expression site, a smaller amount of amplification rounds must be required before reaching a satisfactory level of production. Still further, the subject matter makes possible the reproducible selection of high producing clones wherein the vector is integrated into a low number of copies, typically a single copy. This is advantageous because it improves the stability of the clones and avoids other potential adverse side effects associated with a large number of copies. As described above, the homologous object recombination system uses the combination of a "marker plasmid" and a "target location plasmid", which are described in greater detail below. The "marker plasmid" which is used to label and identify a transcriptional hot spot will comprise at least the following sequences: _ '(i) an ADN region that is heterologous or unique to the mammalian cell genome, which it functions as a source of homology, allows homologous recombination (with a DNA contained in a second plasmid of white). More specifically, the unique region of the DNA (i) will generally comprise a bacterial, viral, yeast, synthetic, or other DNA which is not normally present in the genome of the mammalian cell and the - which additionally does not understand a significant sequence homology or identity with respect to DNA contained in the genome of the mammalian cell.

Essentially, this sequence must be sufficiently "different from a mammalian DNA that will not recombine significantly with the genome of the host cell by means of homologous recombination.The size of such a single DNA will generally be at least about 2 to 10 kilobases of size, or larger, more preferably at least about 10 Kb, as several other investigators have observed an increased frequency of target recombination, when the size of the homology region is increased (Capecchi, Science, 244: 1288- 1292 (1989)).

The upper-bound size of the single DNA which acts as a site "for homologous recombination with a sequence in the second target vector, is dictated broadly by the potential stability constraints (if the DNA is too large it may not be easily integrated on a chromosome and it is difficult to work with very large DNAs, (ii) a DNA that includes a fragment of a selectable marker DNA, typically an exon of a dominant selectable marker gene.The only essential feature of this DNA is that it does not encode a functional selectable marker protein unless it is expressed in association with a sequence contained in the target plasmid Typically, the target plasmid will comprise the remaining exons of the selectable dominant marker gene (those not included in the "plasmid"). location of the target ".) Essentially, a functional selectable marker must be produced only if the homologous recombination (leading to the association and expression of this marker DNA sequence (i) together with the portion (s) of the selectable marker DNA fragment the one (s) which is (are) contained in the target plasmid).

As noted, the current invention exemplifies the use of the neomycin phosphotransferase gene as the dominant selectable marker which is "separated or divided" in the two vectors. However, other selectable markers must also be suitable, for example, the histidinol dehydrogenase gene of Salmonella, the hygromycin phosphotransferase gene, the herpes simplex virus thymidine kinase gene, the adenosine deaminase gene, the glutamine gene synthetase and the phosphoribosyl transferase gene of hypoxanthine-guanine. (iii) a DNA which encodes a functional selectable marker protein, such a selectable marker is different from the selectable marker DNA (ii). This selectable marker provides for the successful selection of mammalian cells in which the marker plasmid is successfully integrated into the cellular DNA. More preferably, it is desirable that the marker plasmid comprises two DNAs of the dominant selectable marker, located at opposite ends of the vector. This is advantageous because it makes it possible for the members to be selected using different selection agents and makes it possible for the additional cells which contain the complete vector to be selected. Additionally, a marker can be a * amplifiable marker to facilitate the amplification of the gene as previously described. Any of the dominant selectable markers in (ii) can be used as well as others generally known in the art. In addition, the marker plasmid may optionally further comprise a rare endonuclease restriction site. This is potentially desirable because this can facilitate segmentation. If present, such a time restriction site should be located close to the middle part of the single region that acts as a substrate for homologous recombination. Preferably such a sequence will be at least about 12 nucleotides. The introduction of a double-strand break by a similar methodology has been reported to improve the frequency of homologous recombination. (Choulika et al, Mol. Cell. Biol., 15: 1968-1973 (1995)). However, the presence of such a sequence is not essential. The "target location plasmid" will comprise at least the following sequences: (1) the same unique region of the DNA contained in the marker plasmid or one having a sufficient homology or sequence identity to it so that the DNA be able to combine by means of homologous recombination with the unique region (i) in the marker plasmid. Suitable types of DNAs are described supra in the description of the unique region of -DNA (1) in the marker plasmid. (2) The remaining exons of the dominant selectable marker, an exon of which is included as (ii) in the marker plasmid listed above. The essential feature of this DNA fragment is that it leads to a functional (selectable) marker protein only if the target plasmid is integrated by means of homologous recombination (where such recombination leads to the association of this DNA with the other fragment of the DNA of the selectable marker Contained in the marker plasmid) and further that it allows the insertion of a desired exogenous DNA. Typically, this DNA will comprise the remaining exons of the selectable marker DNA which are separated by an intron. For example, this DNA can comprise the first two exons of the neo gene and the plasmid of the marker can comprise the third exon (third hind of neo). (3) The target plasmid will also comprise a Desired DNA, for example, one encoding a desired polypeptide, preferably inserted into the DNA fragment of the selectable marker contained in the plasmid. Typically, the DNA will be inserted into the intron which is comprised between the DNA exons of the selectable marker. This ensures that the desired DNA is also integrated if homologous recombination of the target plasmid "and the marker plasmid occurs.This intron may be present naturally or it may be designed in the DNA fragment of the dominant selectable marker. This DNA will encode any desired protein, preferably one that has pharmaceutical properties or other desirable properties.Most typically the DNA will encode a mammalian protein, and in the common examples provided, an immunoglobulin or an immunoadhesin.However, the invention is not limited to no way to the production of immunoglobulins. As previously described, the object cloning method is suitable for many mammalian cells because it does not require the efficacy of any of the specific mammalian sequence or sequences being present. In general, such mammalian cells will comprise those typically used for the expression of the protein, for example, CHO cells, myeloma cells, COS cells, BHK cells, Sp2 / 0 cells, NIH 3T3 and HeLa cells. In the examples that follow, the CHO cells were used. The advantages thereof include the availability of the appropriate growth medium, their ability to grow efficiently and of high density in the culture, and their ability to express mammalian proteins such as immunoglobulins in the biologically active form. In addition, the CHO cells were selected in a large part because of the previous use of such cells by the inventors for the expression of the immunoglobulins (using the vectors containing the dominant marker translationally altered, previously described). Accordingly, the present laboratory has considerable experience in the use of such cells for expression. However, based on the examples that follow, it is reasonable to expect that similar results will be obtained with other mammalian cells. In general, the transformation or transfection of the mammalian cells according to the subject invention will be effected in accordance with conventional methods. So that the invention can be better understood, the construction of the exemplary vectors and their use in the production of members is described in the examples given below.

EXAMPLE 1 Design and Preparation of DNA Vectors Location of Target and Marker The plasmid of the marker referred to herein as "Desmond" will be assembled from the following DNA elements: (a) murine dihydrofolate reductase (DHFR) gene, incorporated in a transcription cassette, comprising the beta 5"promoter mouse globin with respect to the DHFR starting site, and the 3"poly adenylation signal of the bovine growth hormone for the stop codon. The transcriptional cassette of DHFR was isolated from TCAE6, an expression vector previously created in this laboratory (Newman et al, 1992, Biotechnology, 10: 1455-1460). (b) commercially available E. coli β-galactosidase gene, obtained from Promega as the control vector of pSV-β-galactosidase, catalog # E1081. (c) Baculovirus DNA, commercially available, purchased from Clontech as pBAKPAKd, cat. # 6145-1. (d) Enhancers and promoters elements comprising the Cytomegalovirus Casete and the SV40 virus. The cassette was generated by PCR using a derivative of the TCAE8 expression vector (Reff et al, Blood, 83: 435-445 (1994)). The enhancer cassette was inserted into the baculovirus sequence, which was first modified by insertion of a multiple cloning site. (e) GUS (glucuronidase) gene from E. coli, commercially available, purchased from Clontech as PB101, cat. # 6017-1. (f) Firefly luciferase gene, commercially available, obtained from Promega as pGEM-Luc (catalog # E1541). (g) Histidinol dehydrogenase gene of S. typhimurium (HisD). This gene was originally a gift (Donahue et al, Gene, 18: 47-59 (1982)), and has subsequently been incorporated into a transcription cassette comprising the 5 'major promoter of mouse beta globin "for the gene and the 3 'polyadenylation signal of SV40 for the gene The DNA elements described in (a) - (g) were combined in a backbone of the plasmid derived from pBR to produce a contiguous stretch of 7.7kb of the DNA referred to in FIGS. Appended figures as "homology." Homology in this sense refers to DNA sequences which are not part of the mammalian genome and are used to promote homologous recombination between transfected plasmids that share the same DNA sequences of homology (h) TN5 neomycin phosphotransferase gene (Davis and Smith, Ann. Rev .; Micro., 32: 469-518 (1978)). The complete neo gene was "subcloned into pBluescript SK- (Stratagene catalog # '212205) to facilitate genetic manipulation." A synthetic linker was then inserted into a unique Pstl site that is present through the codons for amino acids 51 and 52 from "neo. This linker encoded the necessary DNA elements to create an artificial binding donor site, an intervening intron, and the acceptor site of the junction within the neo gene, thereby creating two separate exons, currently referred to as exons 1 and 2 of neo. The neo exon 1 encodes the "first 51 amino acids of neo, while exon 2 codes for the remaining 203 amino acids plus the stop codon of the cloning site of protein A. The cloning site Notl was also created within the intron." Neon exon 2 was subdivided Additionally .to produce exons 2 and 3 of neo. This was achieved as follows: A set of "PCR primers was designed to • amplify a region of -DNA that encodes exon 1 of "neo, the intron and the first 111 2/3 of the amino acids of exon 2. The primer of 3 'PCR led to the -introduction of a new 5' binding site immediately after the second nucleotide of the codon for the • amino acid 111 in exon 2, thus generating a nine smaller exon 2. The DNA fragment now encodes the original exon 1, the intron and the new exon 2 were subcloned and then propagated in a vector based on pBR. of the original exon 2 was used as a model for another round of "PCR amplification, which quenched the" exon 3". The 5 'primer for this round of amplification introduced a new binding site on the 5' side of newly created exon 3, ie before the final nucleotide of the codon for amino acid 111. The resulting 3 exons of neo encode the following information ": exon 1 - the first 51 amino acids of neo, exon 2 - the following 111 2/3 amino acids, and exon 3 the final 91 1/3 amino acids plus the codon of translational stop of the neo gene. it was incorporated in the company of the aforementioned DNA elements in the "Desmond" tagging plasmid.Neon exons 1 and 2 were incorporated into the target location plasmid "Molly." The Notl cloning site created within the intron between exons 1 and 2 were used in the subsequent cloning steps to insert the genes of interest in the plasmid-target location.

A second target location plasmid "Mandy" was also generated. This plasmid is almost identical to "Molly" (some restriction sites on the vector have been changed) except that the original HisD and DHFR genes contained in "Molly" were inactivated. These changes were incorporated because the Desmond cell line is no longer being cultivated "in the presence of Histidinol, therefore it seems unnecessary to include a second copy of the HisD gene." Additionally, the DHFR gene was inactivated to ensure that only a single DHFR gene, especially the only one present in the Desmond-tagged site, would be amplifiable. in any resulting cell lines. "Mandy" was derived from "Molly" by the following modifications: (i) A synthetic linker was inserted in the middle part of the DHFR coding region, this linker created a stop codon and displaced the rest of the DHFR coding region outside the frame, thereby rendering the gene non-functional (ii) A portion of the HisD gene was deleted and replaced with a fragment of HisD generated by PCR which lacks the promoter and codon of stop of the gene Figure 1 shows the arrangement of these DNA elements in the "Desmond" marker plasmid Figure 2 shows the arrangement of these elements in the first location plasmid n white, "Molly". Figure 3 illustrates the possible arrangement in the CHO genome, of the various DNA elements after the location of the target and the integration of Molly DNA into the CHO cells labeled from Desmond. Figure 9 shows the target location plasmid "Mandy". The construction of the labeling plasmids and target location of the DNA elements listed above was carried out following conventional cloning techniques [see, for example, Molecular Cloning, A Laboratory Manual, J. Sambrook et al, 1987, Cold Spring Harbor Laboratory Press, and Current Protocols in Molecular Biology, FM Ausubel et al, eds., 1987, John Wiley and Sons). All plasmids were propagated and maintained in XLI blue of E. coli (Stratagene, cat # 200236). Large-scale plasmid preparations were prepared using the Wizard Maxiprep® DNA Purification System from Promega, according to the manufacturer's instructions.

EXAMPLE 2 Construction of a CHO Cell Line Marked 1. Transfection and Cell Culture Procedures for the CHO Cell Line Marked, Produced The marker plasmid DNA was linearized by digestion overnight at 37 ° C with Bstll07I. The linearized vector was precipitated with ethanol and resuspended in sterile TE to a concentration of 1 mg / ml. The linearized vector was introduced into Chinese Hamster Ovary (CHO cells) -DHFR cells, DG44 cells (Urlaub et al., Som. Cell and Mol. Gen., 12: 555-566 (1986) by electroporation as follows. exponentially growing cells were collected by centrifugation, washed once in ice-cooled SBS (buffered solution with sucrose, 272 mM sucrose, 7 mM sodium phosphate, pH 7.4, 1 mM magnesium chloride) then resuspended in SBS until a concentration of 107 cells / ml After a 15-minute incubation on ice, 0.4 ml of the suspension of the cells is mixed with 40 μg of the linearized DNA in a disposable electroporation cell. electroceldás manipulator BTX (San Diego, CA) fixed at 230 volts, 400 microfarads of capacitance, 13 ohms of resistance The cells to which the shocks were applied were then mixed with 20 ml of growth medium CHO preheated (CHO-S-SFMII, Gibco / BRL, catalog # 31033-012) and plated in 96-well tissue culture plates. Forty-eight hours after electroporation, the plates were fed with a selection medium (in the case of transfection with Desmond, the selection medium is CHO-S-SFMII, without hypoxanthine or thymidine, supplemented with 2mM Histidinol (Sigma catalog # H6647) .The plates were maintained in a selection medium for up to 30 days, or until some of the cavities exhibited cell growth.These cells were then removed from the 96-well plates and finally expanded to recipients for 120 ml centrifuge where they were kept in the selection medium all the time. ~ EXAMPLE 3 Characterization of Marked CHO Cell Lines (a) Southern analysis Genomic DNA was isolated from the Desmond-labeled CHO cells that all grow stably. The DNA was isolated using the set or set Invitrogen Easy®, according to the manufacturer's instructions. The genomic DNA was then digested with HindIII overnight at 37 ° C, and subjected to Southern analysis using a digoxigenin labeled probe generated by PCR, specific for the DHFR gene. Hybridizations and washes were carried out using DIG Easy Hyb from Boehringer Mannheim (catalog # 1603 558) and the DIG Wash and Block Buffer Set (catalog # 1585 762) according to the manufacturer's instructions. DNA samples that contain a single band that hybridizes to the DHFR probe are supposed to be clones of Desmond that arise from a single cell which has integrated a single copy of the plasmid. These clones were retained for further analysis. Out of a total of 45 isolated HisD-resistant cell lines, only 5 were members of a single copy. Figure 4 shows a Southern spotting containing all of 5 of these single-copy Desmond clones. The names of the clones are provided in the legend of the Figures. (b) Northern analysis Total RNA was isolated from all Desmond clones in a single copy using the TRIzol reagent (Gibco / BRL cat. # 15596-026) according to the manufacturer's instructions. 10-20 μg of the RNA of each clone were analyzed on duplicate formaldehyde gels. The resulting spots were probed with DNA probes labeled with digoxigenin generated by PCR for (i) the DHFR message, (ii) the HisD message and (iii) the CAD message. CAD is a trifunctional protein involved in the biosynthesis of uridine (Wahl et al, J. Biol. Chem., 254, 17: 8679-8689 (1979)), and is also expressed in all cell types. It is used here as an internal control to help load the candidate RNA. Hybridizations and washings were carried out using the reagents from Boehringer Mannheim mentioned above. The results of the Northern analysis are shown in Figure 5. The single-copy Desmond clone exhibiting the highest levels of both His D and the DHFR message is clone 15C9, shown in band 4 in both panels of the Figure. This clone was designated as the "marked cell line" and was used in future target placement experiments in CHO, examples of which are presented in the following sections.

EXAMPLE 4 Expression of Anti-CD20 Antibody in CHO cells Marked by Desmond C2B8, a chimeric antibody which recognizes the antigen on the surface of CD20 B cell, has been cloned and previously expressed in the laboratory of the inventors. (Reff et al, Blood, 83: 434-45 (1994)). A DNA fragment of 4.1 kb comprising the light and heavy chain genes of C2B8, in the company of the necessary regulatory elements (the eukaryotic promoter and the polyadenylation signals) was inserted into the artificial intron created between exons 1 and 2 of the neo gene contained in a cloning vector derived from pBR. This recently generated 5kb DNA fragment (comprising neo exon 1, C2B8 and neo exon 2) was excised and used to assemble the Molly target location plasmid. The other DNA elements used in the construction of Molly are identical to those used to construct the Desmond labeling plasmid, previously identified. A complete map of Molly is shown in Figure 2. The Molly target location vector was linearized prior to transfection by digestion with Kpnl and Pací, precipitated in ethanol and resuspended in sterile TE at a concentration of 1.5 mg / ml. The linearized plasmid was introduced into the Desmond-labeled cells that grow exponentially, essentially as described, except that 80 μg of the DNA was used in each electroporation. Forty-eight hours post-electroporation, 96-well plates were supplemented with the selection medium - CHO-SSFMII supplemented with 400 μg / ml Geneticin (G418, Gibco / BRL catalog # 10131-019). The plates were kept in the selection medium for up to 30 days, or until the growth of the cells occurred in some of the cavities. Such growth was assumed to be the result of the clonal expansion of a single G418 resistant cell. Supernatants from all G418 resistant cavities were evaluated to verify the production of C2B8 by standard ELISA techniques, and all productive clones were eventually expanded to 120 ml centrifuge vessels and further analyzed.

Characterization of Target or Target Cells that secrete the Antibody A total of 50 electroporations with the Molly target location plasmid were carried out in this experiment, each of which was plated in boxes of 96 separate cavities. A total of 10 clones secreting anti-DC-20 antibodies were obtained and expanded into 120 ml centrifuge containers. Genomic DNA was isolated from all the clones, and Southern analyzes were subsequently performed to determine whether the clones represented unique homologous recombination events or whether additional random integrations have occurred in the same cells. The methods for DNA isolation and Southern hybridization were as described in the previous section. The genomic DNA was digested with EcoRI and probed with a probe labeled with digoxigenin generated by PCR to a segment of the constant region of the heavy chain of CD20. The results of this Southern analysis are presented in Figure 6. As can be seen in the Figure, 8 of the 10 clones show a single band that hybridizes to the CD20 probe, indicating that a single homologous recombination event has occurred in these cells. Two of the ten clones, clones 24G2 and 28C9, r show the presence of additional band (s), indicative of an additional random integration in any other part of the genome. The expression levels of the anti-CD20 antibody were examined in all of these ten clones, the data of which are shown in the Table 1, later.

Table 1: Level of Expression of the Homologous Members that Secrete the Ant? -CD20 Clone Anti-CD20, pg / c / d 20F4 3.5 25E1 2.4 42F9 1.8 39G11 1.5 21C7 1.3 50G10 0.9 29F9 0.8 5F9 0.3 28C9 * 4.5 24G2 * 2.1 • These clones contained additional randomized copies of the anti-CD20. The expression levels of these clones therefore reflect a contribution of both homologous and random sites.

Expression levels are reported as picogram per cell per day (pg / c / d) secreted by individual clones, and represented the average levels obtained from three separate ELISAs on samples taken from 120 ml centrifuge vessels. As can be seen from the data, there is a variation in the secretion of the antibodies approximately ten times between the highest and lowest clones. This was unexpected because similar expression levels of all the clones were anticipated due to the fact that the anti-CD20 genes are all integrated in the same marked site of Desmond. However, this range observed in the expression is extremely small compared to that observed using any traditional random integration method or without the translationally altered vector system of the invention. The clone 20F4, the member of a single higher production copy, was selected for further study. Table 2 (below) presents the cell culture and ELISA data of the seven-day production runs of this clone.

Table 2: Data of the Run of the Production of 7 Days for 20F4 Day% Available- Available / TX2 (h) mg / l pg / c / d nible ml (x 10 ~ r "1 96 3.4 31 1.3 4.9 2 94 6 29 2.5 3.4 3 94 9.9 33 4.7 3.2 4 90 17.4 30 6.8 3 5 73 14 8.3 6 17 3.5 9.5 The clone 20F4 was seeded in 2xl05 ml in a 120 ml centrifuge container on day 0. In the following six days, the cell counts were taken, the calculated times and samples of the 1 ml supernatant were removed from the container and analyzed to check the anti-Cd20 secreted by ELISA.

This clone is secreting on average, 3-5 pg antibody / cell / day, based on these ELISA data. This is the same level as that obtained from other clones of a single high expression copy, previously obtained in the inventors' laboratory using the randomly altered randomization vectors previously developed. This result indicates the following: (1) that the site in the CHO genome labeled by the Desmond labeling vector is highly transcriptionally active, and pdr "represents both an excellent site from which recombinant proteins can be expressed, and (2) that the location as a target by means of homologous recombination can be done using the object vectors and occurs at a sufficiently high frequency to make this system a viable and desirable alternative for the methods of random integration. In addition to the effectiveness of this system, this site has been shown to be amplifiable, leading to even higher levels of gene expression and protein secretion Amplification is achieved by plating serial dilutions of 20F4 cells , starting from a density of 2.5 x 104 cells / ml, in discs or tissue culture plates of 96 cavities, and cultivating these cells in the middle (CHO-SSFMII) supplemented with 5, 10, 15 or 20 nM of methotrexate. The secretion clones of the antibody were selected using standard ELISA techniques, and the higher production clones were expanded and analyzed further. A summary of this amplification experiment is presented in Table 3 below.

Table 3: Summary of the Amplification of 20F4 nM MTX # Cavities Expression Level # Expression Level Cavities Tested mg / l 96 cavities Expanded pg / c / d from the centrifuge 56 3-13 10-15 15 27 2-14 15-18 17 4-11 ND The amplification of the methotrexate of 20F4 was established as described in the text, using the concentrations of methotrexate indicated in the table above. Supernatants from all colonies of 96 surviving cavities were evaluated by ELISA, and the range of ant? -CD20 expressed by these clones is indicated in column 3. Based on these results, the highest production clones were expanded to 120 ml containers for centrifugation and several ELISAs were carried out on the supernatants of the centrifuge to determine the expression levels of pg / cell / day, reported in column 5.

The aqui data clearly demonstrates that this site can be amplified in the presence of methotrexate. Clones of 10 and 15 nM amplifications were found to produce on the order of 15-20 pg / cell / day. A 15 nM clone, designated 20F4-15A5, was selected as the highest expression cell line. This clone originated from a plate of 96 cavities in which there was only growth in 22 cavities, and therefore it is assumed that they have emerged from a single cell. A 15nM clone, designated 20F4-15A5, was selected as the highest expression cell line. This clone originated from a plate of 96 cavities in which there was only growth in 22 cavities, and therefore it is assumed that they have emerged from a single cell. The clone was then subjected to an additional round of methotrexate amplification. How I know -described above, the serial dilutions of the culture were placed ^ in plates, in boxes of 96 cavities and cultured in a medium of CHO-SS-FMII supplemented with 200, 300 or 400 nM of methotrexate. The surviving clones were selected by ELISA, and several high production clones were expanded to cultures in centrifuge machine containers and further analyzed. A summary of this second amplification experiment is presented in Table 4.

Table 4: Summary of the Amplification of 20F4-15A5 nM MTX # Cavities Expression Level # Expression Level Cavities Tested mg / l 96 cavities Expanded pg / c / d, from the centrifuge 200 67 23-70 50-60 250 21-70 55-60 300 15-75 40-50 The methotrexate amplification of 20F4-15A5 was established and evaluated as described in the text. The highest production cavities, the numbers of which are indicated in column 4, were expanded into containers for a 120 ml centrifuge machine. The expression levels of cell lines derived from these cavities are recorded as pg / c / d in column 5.

The highest production clone came from the amplification of 250 nm methotrexate. The 250 nM clone, 20F4-15A5-250A6, originated from a 96-well plate in which only in some cavities there was growth, and therefore it is assumed that they have arisen from a single cell. Taken together, the data in Tables 3 and 4 strongly indicate that two rounds of methotrexate amplification are sufficient to reach expression levels of 60 pg / cell / day, which is approximately at the maximum secretion capacity of the immunoglobulin in mammalian cells (Reff, ME, Curr Opin, Biotech., 4: 573-576 (1993)). The ability to achieve this secretory capacity with only two steps of amplification further improves the usefulness of this homologous recombination system. Typically, random integration methods require more than two steps of amplification to reach the level of expression and are generally less reliable in terms of ease of amplification. Accordingly, the homologous system offers a time-saving and more efficient method for achieving high-level gene expression in mammalian cells.

EXAMPLE 5 Expression of Anti-Human CD-23 Antibody in Desmond's CHO Cells CD-23 is a low affinity IgE receptor which mediates the binding of IgE to B and T lymphocytes (Sutton, B.J., and Gould, H.J., Nature, 366: 421-428 (1993)). The monoclonal antibody of anti-human CD23 5E8 is a human monoclonal gamma-1 cloned recently and expressed in the laboratory of the inventors. This antibody is described in commonly assigned Serial No. 08 / 803,085, filed on February 20, 1997. The light and heavy chain genes of 5E8 were cloned into the expression vector of the mammal N5KG1, a derivative of the NEOSPLA vector (Barnett et al, in Antibody Expression and Engineering, HY Yang and T. Imanaka, eds., pp. 27-40 (1995) and two modifications were then made to the genes.) A slightly more secretion has recently been observed. Elevation of immunoglobulin light chains compared to heavy chains in other expression constructs in the laboratory (Reff et al., 1997, unpublished observations). In an attempt to compensate for this deficiency, the heavy chain gene of 5E8 has been altered by the addition of a stronger promoter / enhancer element immediately upstream of the starting site. In the subsequent steps, a 2.9 kb DNA fragment comprising the light and heavy chain genes, modified from 5E8, was isolated from the N5KG1 vector and inserted into the Mandy target location vector. The preparation of Molly containing 5E8 and the electroporation in the CHO 15C9 cells of Desmond was essentially as described in the preceding section. A modification to the previously described protocol was in the type of culture medium used. Desmond-labeled CHO cells were cultured in a protein-free CD-CHO medium (Gibco-BRL, catalog # AS21206) supplemented with 3 mg / l recombinant insulin (3 mg / ml storage material, Gibco-BRL , catalog # AS22057) and 8 mM of L-glutamine (200 inM of the raw material, Gibco-BRL, catalog # 25030-081). Subsequently, the transfected cells were selected in the above-supplemented medium with 400 μg / ml of geneticin. In this experiment, 20 electroporations were made and plated in tissue culture boxes of 96 wells. The cells grew and secreted the anti-CD23 in a total of 68 cavities, all of which were assumed to be clones originating from a single G418 cell.Twelve of these cavities were expanded into centrifuge machine containers. of 120 ml for further analysis It is believed that the increased number of clones isolated in this experiment (68 compared to 10 for the anti-CD20 as described in Example 4) is due to a cloning efficiency and at a rate of Higher survival of the cedulas that have grown in a CD-CHO medium compared to the medium of CHO-SS-FMII The expression levels for these clones analyzed in the culture of the centrifugal machine varied from 0.5-3 pg / c / d, in full agreement with the levels observed for the anti-CD20 clones The high production anti-CD23 clone, designated 4H12, was subjected to amplification of methotrexate to increase its expression levels. established in a manner similar to that described for the anti-CD20 clone in example 4. The serial dilutions of the exponentially growing 4H12 cells were plated in tissue culture boxes of 96 wells and grown in a medium of CD-CHO supplemented with 3 mg / l of insulin, 8 mM of glutamine and 30, 35 or 40 nM of methotrexate. A summary of this amplification experiment is presented in Table 5.

Table 5: Summary of the Amplification of 2H12 nM MTX # Cavities Expression Level # Expression Level Cavities Tested mg / l 96 cavities Expanded pg / c / d from the centrifuge 100 6 = 2 10-25 64 4-27 10-15 40 96 4-20 ND The highest expression clone obtained, was a clone of 30 nM, isolated from a plate on which in 22 cavities there has been growth. This clone, designated 4H12-30G5, was reproducibly secreting 18-22 pg of antibody per cell per day. This is the same range of expression observed for the first amplification of anti-CD20 clone 20F4 (clone 20F4-15A5 which produced 15-18 pg / c / d, as described in Example 4). These data serve to additionally support the observation that amplification at this CHO-labeled site is reproducible and efficient. A second amplification of this 30 nM cell line is usually in motion. From this it follows that the saturation levels of the expression will be achievable for the anti-CD23 antibody in only two steps of amplification, as was the case for the ant? -CD20. EXAMPLE 6 - Expression of Immunoadhesin in CHO cells Marked by Desmond CTLA-4, an element of the Ig superfamily, is found on the surface of T lymphocytes and is thought to play a role in the activation of the T cell specific for Zel antigen (Dariavach et al, Eur. J. Immunol ., 18: 1901-1905 (1988), and Linsley et al, J. Exp. Med., 174: 561-569 (1991)). To further study the precise role of the CTLA-4 molecule in the activation pathway, a soluble fusion protein comprising the extracellular domain of CTLA-4 bound to a truncated form of the constant region of human IgGl was created I (Linsley et al (Id.) The inventors have recently expressed this CTLA-4 Ig fusion protein in the mammalian expression vector BLECH1, a derivative of the NEOSPLA plasmid (Barnett et al, in Antibody Expression and Engineering, H.Y. Yang and T. Imanaka, eds., Pp. 27-40 (1995)). An 800 bp fragment encoding the CTLA-4 Ig was isolated from this vector and inserted between the SacII and BglII sites in Molly. Preparation of the CTLA-4Ig-Molly and electroporation in the CHO cells of the Desmond clone 15C9 was carried out as described in the previous example which refers to the anti-CD20. Twenty electroporations were carried out, and plated in 96-well culture boxes as previously described. Eighteen cavities expressing the CTLA-4 were isolated from the 96-well plates and carried forward to the centrifuge stage in 120 ml containers. Southern analyzes on the isolated genomic DNA of each of these clones were carried out to determine how many of the homologous clones contained additional random members. The genomic DNA was digested with BglII and probed with a probe labeled with digoxigenin generated by PCR with respect to the constant region of human IgGl. The results of this analysis indicated that 85% of CTLA-4 clones are homologous members only; the remaining 15% contained an additional random member. This result corroborates the discoveries of the anti-CD20 expression described above, where 80% of the clones were unique homologous members. Therefore, it can be concluded that this expression system reproducibly produces homologous members located as a target, unique, in at least 80% of all the produced clones. The expression levels for the homologous CT1A4-Ig clones ranged from 8-12 pg / cell / day. This is somewhat higher than the range reported for the anti-CD20 and anti-CD23 antibody clones described above. However, it has been previously observed that the expression of this molecule using the intronic insertion vector system also led to significantly higher expression levels than those obtained for immunoglobulins. Currently it has not been possible to provide an explanation for this observation.

EXAMPLE 7 Location of an Anti-CD20 White for a CHO Cell Line marked by Desmond As described in a preceding section, 5 CHO cell lines labeled from Desmond were obtained from a single copy (see Figures 4 and 5). To 'demonstrate that the success of the location strategy as a target of the invention is not due to any unique property of the Desmond clone 15C9 and was limited only to this clone, the anti-CD20 Molly was introduced into the clone 9B2 of Desmond (Strip 6 in Figure 4, strip 1 in Figure 5). The preparation of the Molly DNA and the electroporation in Desmond 9B2 was exactly as described in the previous example belonging to the anti-CD20. A homologous member of this experiment was obtained. This clone was expanded to a 120 ml centrifuge machine vessel, where it produced on average 1.2 pg of the anti-CD20 / cell / day. This is a considerably lower expression than that observed with the Molly located as white in Desmond 15C9. However, this was the anticipated result, based on our northern analysis of the Desmond clones. As can be seen in Figure 5, the mRNA levels of clone 9B2 are considerably lower than those of 15C9, indicating the site in this clone that is not transcriptionally active as that in 15C9. Therefore, this experiment not only demonstrates the reproducibility of the system - presumably any marked Desmond site can be targeted as Molly - but also confirms the northern data that the site on Desmond 15C9 is the most transcriptionally active site.

From the foregoing, it will be appreciated that, although the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the scope of the invention. Accordingly, the invention is not limited to the appended claims.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (41)

1. A method for inserting a desired DNA into a target site in the genome of a mammalian cell, characterized in that it comprises the following steps: (i) transfecting or transforming a mammalian cell with a first plasmid ("tag plasmid") containing the following sequences: (a) a region of DNA that is heterologous to the genome of the mammalian cell which when integrated into the genome of the mammalian cell provides a unique site for homologous recombination; (b) a DNA fragment encoding a portion of a first selectable marker protein; and (c) at least one other selectable marker DNA that provides for the selection of mammalian cells which have been successfully integrated with the marker plasmid; (ii) selecting a cell which contains the marker plasmid integrated into its genome; (iii) transfecting or transforming the selected cell with a second plasmid ("blank plasmid") which contains the following sequences: (a) a region of DNA that is identical or is sufficiently homologous with respect to the single region in the plasmid marker such that this region of the DNA can recombine with such DNA by means of homologous recombination; (b) a DNA fragment encoding a portion of the same selectable marker contained in the marker plasmid, wherein the active, selectable marker protein encoded by the DNA is produced only if the fragment is expressed in association with the fragment of the DNA of the selectable marker contained in the marker plasmid; and (iv) selecting the cells which contain the target plasmid integrated in the target site by selection or screening for the expression of the first marker protein.

2. The method according to claim 1, characterized in that the fragment of DNA encoding a fragment of a first selectable marker is an exon of a dominant selectable marker.

3. The method according to claim 2, characterized in that the second plasmid contains the remaining exons of the first selectable marker.

4. The method according to claim 3, characterized in that at least one DNA encoding a desired protein is inserted between the exons of the first selectable marker contained in the target or target plasmid.

5. The method according to claim 4, characterized in that a DNA encoding a dominant selectable marker is additionally inserted between the exons of the first selectable marker contained in the target or target plasmid to provide co-amplification of the DNA encoding the desired protein.

6. The method according to claim 3, characterized in that the first dominant selectable marker is selected from the group consisting of neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, thymidine kinase of herpes simplex virus, adenosine deaminase, glutamine. synthetase, and hypoxanthine phosphoribosyl transferase- "guanine.

7. The method according to claim 4, characterized in that the desired protein is a mammalian protein.

8. The method according to claim 7, characterized in that the protein is an immunoglobulin. _

9. The method according to claim 1, characterized in that it further comprises determining the levels of 7? JRN of the selectable marker (c) contained in the marker plasmid prior to the integration of the targeting vector.

10. The method according to claim 9, characterized in that the other selectable marker contained in the marker plasmid is a dominant selectable marker selected from the group consisting of histidinol dehydrogenase, herpes simplex thymidine kinase, hydromycin phosphotransferase, adenosine deaminase and glutamine. synthetase

11. The method according to claim 1, characterized in that the mammalian cell is selected from the group consisting of cells from the Chinese hamster ovary (CH), myeloma cells, baby hamster kidney cells, COS cells, cells HeLa and NIH 3T3 cells.

12. The method according to claim 11, characterized in that the cell is a --- CHO cell.

13. The method according to claim 1, characterized in that the marker plasmid contains the third exon of the neomycin phosphotransferase gene and the target or target plasmid contains the first two exons of the neomycin phosphotransferase gene.

14. The method according to claim 1, characterized in that the marker plasmid also contains a rare restriction endonuclease sequence which is inserted within the region of homology.

15. The method according to claim 1, characterized in that the unique region of the DNA that is provided for the homologous recombination is a bacterial DNA, a viral DNA or a synthetic DNA.

16. The method according to claim 1, characterized in that the single region of the DNA that is provided for the homologous recombination is at least 300 nucleotides.

17. The method according to claim 16, characterized in that the single region of the DNA varies in size from about 300 nucleotides to 20 kilobases.

18. The method according to claim 17, characterized in that the single region of the DNA preferably varies in size from 2 to 10 kilobases.

19. The method according to claim 1, characterized in that the first DNA of the selectable marker is divided into at least three -exons.

20. The method according to claim 1, characterized in that the single region of the DNA that is provided for the homologous recombination is a bacterial DNA, an insect DNA, a viral DNA or a synthetic DNA.

21. The method according to claim 20, characterized in that the single region of the DNA does not contain any "functional gene".

22. A system of vectors for inserting a desired DNA into a target or target site in the genome of a mammalian cell, characterized in that it comprises at least the following: (i) a first plasmid ("tag plasmid") containing the minus the following sequences: (a) at least one region of DNA that is heterologous with respect to the genome of the mammalian cell, which when integrated into the genome of the mammalian cell provides a unique site for homologous fecombination; (b) a DNA fragment encoding a portion of a first selectable marker protein; and (c) at least one other DNA of the selectable marker that is provided for the selection of mammalian cells which have been successfully integrated with the marker plasmid; and ^ (ii) a second plasmid ("target or target plasmid") which contains at least the following sequences: (a) a region of the DNA that is identical or is sufficiently homologous with respect to the unique region in the plasmid of the marker in such a way that this region of the DNA can recombine with the DNA by means of homologous recombination; (b) a DNA fragment encoding a portion of the same selectable marker contained in the marker plasmid, wherein the active selectable marker protein encoded by the DNA is produced only if the fragment is expressed in association with the marker DNA fragment selectable content in the marker plasmid.

23. The vector system according to claim 22, characterized in that the DNA fragment encoding a fragment of a first selectable marker is an exon of a dominant selectable marker.

24. The vector system according to claim 23, characterized in that the second plasmid contains the remaining exons of the first selectable marker.

25. The vector system according to claim 24, characterized in that at least one DNA encoding a desired protein is inserted between the exons of the first selectable marker contained in the target or target plasmid.

26. The vector system according to claim 24, characterized in that a DNA encoding a dominant selectable marker is additionally inserted between the exons of the first selectable marker contained in the target or target plasmid to provide co-amplification of the DNA encoding the desired protein .

27. The vector system according to claim 24, characterized in that the first dominant selectable marker is selected from the group consisting of neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and phosphoribosyl transferase of hypoxanthine-guanine.

28. The vector system according to claim 25, characterized in that the desired protein is a mammalian protein.

29. The vector system according to claim 28, characterized in that the protein is "an immunoglobulin.

30. The vector system according to claim 22, characterized in that the other selectable marker contained in the marker plasmid is a dominant selectable marker selected from the group consisting of histidinol dehydrogenase, herpes simplex thymidine kinase, "hydromycin phosphotransferase, adenosine deaminase and glutamine synthetase.

31. The vector system according to claim 22, characterized in that it is provided for the insertion of a desired DNA into a target or target site in the genome of a mammalian cell selected from the group consisting of ovarian cells of the Chinese hamster ( CHO), myeloma cells, baby hamster kidney cells, COS cells, NSO cells, HeLa cells and NIH 3T3 cells.

32. The vector system according to claim 31, characterized in that the mammalian cell is a CHO cell.

33. The vector system according to claim 22, characterized in that the marker plasmid contains the third exon of the neomycin phosphotransferase gene and the target or target plasmid contains the first two exons of the neomycin phosphotransferase gene.

3 . The vector system according to claim 22, characterized in that the marker plasmid also contains a rare restriction endonuclease sequence which is inserted "within the region of homology.

35. The vector system according to claim 22, characterized in that the single region of the DNA that is provided for the homologous recombination is a bacterial DNA, a viral DNA or a synthetic DNA.

36. The vector system according to claim 22, characterized in that the single region of the DNA (a) contained in the vector system of the marker plasmid which is provided for the homologous recombination is at least 300 nucleotides.

37. The vector system according to claim 36, characterized in that the single region of the DNA varies in size from about 300 nucleotides to 20 kilobases.

38. The vector system according to claim 37, characterized in that the single region of the DNA preferably varies in size from 2 to 10 kilobases.

39. The vector system according to claim 22, characterized in that the first selectable marker DNA is separated into at least three ioons.

40. The vector system according to claim 22, characterized in that the single region of the DNA that is provided for the homologous recombination is a bacterial DNA, an insect DNA, a viral DNA or a synthetic DNA.

41. The vector system according to claim 40, characterized in that the single region of the DNA does not contain any functional gene.