HK1081595B - Method of expressing recombinant protein in cho cells - Google Patents

Description

Method for expressing recombinant protein in CHO cell

The present invention relates to methods for expressing recombinant product genes in CHO cell lines, to recombinant CHO cell lines, and to novel expression vector constructs.

Chinese Hamster Ovary (CHO) mammalian expression systems are widely used for the production of recombinant proteins. In addition to lymphocyte cell lines such as hybridoma cell lines, it is one of the few cell lines that can be easily and efficiently cultured in high-density animal cell suspension. Moreover, they have a very high yield and a strong resistance to metabolic stress, and it is difficult to culture lymphocytes on an industrial scale. This is crucial to obtain the maximum yield of protein per run of bioreactor, in view of production costs. The choice of the composition of the medium, the design and operation of the bioreactor, these parameters influence the yield, but their optimization is very complex. Of promoters which enhance the regulation of protein expressionThe strength and transcriptional activity are more likely to increase the yield. During the rest period at 10⁶To 10⁷In high density or fed batch cultures where the gene expression is carried out at cell density per ml, an increase in the expression level of a single cell will translate into a considerable increase in yield.

US5,866,359 describes an enhancement of the expression of the already strong hCMV promoter by co-expression of a weak promoter with adenovirus E1A in CHO cells and NSO cells. E1A is a multifunctional transcription factor acting on cell cycle regulation and having independent transcription activation domain and repression function domain. Fine-tuning the expression level of E1A to lower levels is key to achieving an ideal balance between gene transactivation and avoiding adverse effects on cell cycle progression. In addition to the need for careful selection of promoter-driven expression of E1A, a disadvantage of this system is that it partially inhibits the protein synthesis capacity of E1A expressing cells rather than expression of the recombinant protein of interest.

WO95/17516 uses the murine immunoglobulin gamma 2A locus to direct expression vector constructs to highly active loci in lymphocytes of the B cell line, such as the widely used NSO myeloma. The NSO cell is a murine plasma cell or B cell tumor cell line. Only in B cells, but also the chromatin of the immunoglobulin loci is in a fully activated and open state, is it allowed for the natural immunoglobulin promoters or recombinant expression constructs integrated in these loci to exhibit high transcriptional activity.

The disadvantage is that, due to the principle of homologous recombination, the targeting sequence can only be effectively targeted in murine cell lines that match the gamma 2A targeting sequence containing the recombination hot spot; to achieve high levels of expression, the γ 2A locus region must be a gene transcription active region, which limits the effectiveness of the targeting sequence for homologous recombination with B cell-like cells.

The object of the present invention is to provide an expression system suitable for the expression of CHO proteins, which system is capable of enhancing the expression of standard promoters. According to the invention, the object was surprisingly achieved by equipping the expression vectors of CHO cells with a gene targeting sequence which was originally used for homologous recombination in murine B cells.

The drawings show several possible embodiments of the invention. Wherein:

FIG. 1: during transient transfection of CHO-K1 cells, the hCMV promoter and the hCMV promoter were expressed at relative levels of Green Fluorescent Protein (GFP) in the presence of the IgG 2A hotspot sequence.

FIG. 2: relative expression levels of GFP in stably transfected CHO-K1 cells, hCMV promoter and hCMV promoter in the presence of IgG 2A hot spot sequence.

FIG. 3: plasmid map of hCMV-MIE expression vector with IgG 2A targeting sequence.

According to the present invention, a DNA sequence for expressing a recombinant gene in a mammalian cell comprises a recombinant product gene and a promoter for regulating the expression of the product gene, preferably a CMV promoter, and further comprises a mouse immunoglobulin gamma 2A locus DNA sequence or a fragment thereof or a sequence variant thereof capable of enhancing the expression regulated by the promoter. According to the invention, such DNA sequences can be used for constructing expression vectors for expressing recombinant products in CHO cells.

According to the invention, the expression of the recombinant protein comprises the following steps:

a. culturing a CHO cell transfected with an expression vector containing a promoter capable of driving expression of a recombinant protein in the CHO cell and a murine IgG 2A locus DNA or a sequence variant or fragment thereof capable of enhancing the activity of said promoter, and

b. and (5) harvesting the product protein.

Recombinant products of the invention refer to product proteins that are desired to be expressed and harvested in large quantities. It can be a subunit of a variety of proteins of interest, e.g., therapeutic proteins such as interleukins, multimeric proteins such as enzymes, or antibodies, or fragments thereof. The recombinant product gene may comprise a signal sequence encoding a sequence which causes the expressed polypeptide to be secreted extracellularly from the host producer cell. In another preferred embodiment of the invention, the product protein is a secreted protein. More preferably, the first protein or product protein is an antibody or engineered antibody or fragment thereof, preferably an immunoglobulin G (IgG) antibody.

The DNA sequence of the murine immunoglobulin gamma 2A locus (IgG 2A) was originally designed in WO95/17516 as a genome targeting sequence for the production of stable recombinant B lymphocyte cell lines with highly expressed recombinant products. B lymphocytes or plasma cells typically express immunoglobulin RNA of the Ig heavy chain locus at very high levels, probably because of its cell-type specific promoter/transcription factor activity and open chromatin structure. Preferred murine immunoglobulin gamma 2A gene sequences of the present invention are identical to the targeting sequences used in WO 95/17516. This is a 5.1kb BamHI genomic fragment comprising the entire murine Ig γ 2A coding region except for the most 5' portion of the CH1 exon (Yamawaki-Kataoka, Y. et al, proceedings of the American academy of sciences (1982) 79: 2623-containing 2627; Hall, B. et al, molecular immunology (1989) 26: 819. quadrature.826; Yamawaki-Kataoka, Y. et al, nucleic acids Res (1981) 9: 1365-containing 1381). According to the present invention, promoting site-directed homologous recombination is not a relevant immunoglobulin gamma 2A (IgG 2A) gene trait. Thus, variants of the IgG 2A gene sequence, or fragments of variants thereof, are within the scope of the invention if expression of the recombinant product gene under the control of a promoter, such as the preferred hCMV promoter, can be enhanced under transient or stable expression conditions in CHO cells.

Thus, "functional" variants include, for example, insertions, deletions or point mutations of bases and can be generated by methods well known in the art, for example, primer-guided PCR, "error-prone" PCR, "gene-shuffling" PCR, i.e., by PCR recombination of overlapping DNA fragments, or by random mutagenesis in vivo of bacterial clones followed by gene bank transfection and functional selection in CHO cells. For example, random mutagenesis can be carried out with alkylating agents or UV-irradiation as described in molecular genetics experiments (Cold spring harbor, 1972) of Miller, J. Alternatively, native mutants of the host bacteria may also be employed.

Such variant sequences or sequence fragments have a sequence homology of at least 65%, preferably at least 75%, most preferably at least 90% to the corresponding part of the native immunoglobulin gamma 2A locus in the DNA sequence. For example, a SalI restriction site can be inserted into the native StuI site 39bp upstream of membrane exon 2(M2) as a unique linearization site within the murine immunoglobulin γ 2A sequence; such sequence variants were originally designed for site-directed recombination targeting, but may also be used for the purposes of the present invention.

"promoter" refers to a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. The promoter of the present invention is a promoter active in CHO cells. Such a promoter is preferably a strong promoter. A strong promoter refers to a promoter that induces mRNA synthesis in CHO-KI cells at a frequency equal to or higher than the hCMV core promoter/enhancer fragment (US 5168062). Such a promoter may be a cell type dependent promoter as described, for example, in US5589392, but more preferably a universal strong promoter, especially a constitutively active viral promoter, for example the early or late promoter of SV40 virus, the immediate early promoter of human or murine cytomegalovirus (mCMV or hCMV), the Thymidine Kinase (TK) promoter of herpes simplex virus, the Rous sarcoma virus long terminal repeat promoter (RS-LTR), especially the hCMV-MIE promoter in the 2.1kb PstI fragment as described in US5,385,839 and/or EP-323997-A1. The hCMV promoter described in EP-323997-A1, which carries the entire first functional intron of the hCMV Major Immediate Early (MIE) gene, is particularly preferred in embodiments of the present invention.

The hCMV promoter used in the present invention preferably has no "regulator" sequence portion in its upstream/enhancer portion. It has been found that the "regulator" sequence, which extends from-750 bp to-1150 bp from the MIE transcription start site (Meier et al, 1996, Intervirology 39: 331-342, "regulation of hCMV immediate early gene expression"), is particularly disadvantageous in transient transfections, is detrimental to the activity of the hCMV promoter in CHO cells. The enhancement of the hCMV promoter by IgG 2A hot-spot sequences without regulator sequences (without regulators or abbreviated mod-) is even more pronounced.

Transient transfection is characterized by the absence of any selective pressure on the selectable marker carried by the vector. Transient transfection results in a mixed population of cells in which there are cells that have taken up and are capable of expression of the exogenous DNA and cells that have not taken up the exogenous DNA. Those cells expressing the exogenous expression cassette often do not have the transfected DNA integrated into the genome and are therefore prone to loss of exogenous DNA, and their growth in culture of the transiently transfected cell population is superior to that of the transfected cells. Therefore, expression was strongest immediately after transfection and then gradually decreased. Preferably, the transient transfectants described herein are understood to be cells that are capable of being maintained in cell culture for 90 hours after transfection, in the absence of selective pressure.

Preferably, the transfected CHO host cells of the invention are stably transfected cells, especially in respect of the hCMV promoter described previously. Stable transfection, i.e. the newly introduced foreign DNA is integrated into the genomic DNA by random non-homologous recombination; in the case of vector sequences, the stable transfection described in the present invention may lose part of the vector sequence not directly associated with the gene expression of the recombinant product, for example as an unwanted bacterial copy number control region after genomic integration. The transfected cell has integrated into its genome at least a portion of the expression vector or a plurality of different portions. Similarly, the transfected host cells of the invention also include CHO cells transfected with two or more DNA sequences to form at least functional equivalents of the essential elements of the expression vectors of the invention in vivo, i.e., a suitable promoter and the product gene under the control of the IgG 2A hotspot sequence. For the in vivo assembly of functional DNA sequences after transfection of DNA fragments see, for example, WO 99/53046. Such stable integration may result in the formation of double minichromosomes in CHO cells if further subjected to selective pressure applied for gene amplification. This also pertains to "stabilization" as described herein. After the expression vector is randomly integrated into a CHO cell genome, the guide sequence can enhance the activity of the promoter for promoting the expression of the recombinant product protein. This effect cannot be achieved or predicted based on homologous gene targeting in mature murine B cell lines such as plasmacytoma (plasmacytoma) myeloma cell lines; in that orientation, the role of the IgG 2A targeting sequence is limited to increasing the frequency of high yield homologous integrants (integrants), since the IgG 2A locus is a recombination "hot spot". As described above, chromatin of an immunoglobulin genomic region is in an open, highly active state within a suitable target B cell.

"expression vector" refers to a DNA sequence necessary for transcription and translation of mRNA in a suitable mammalian host cell following transfection. A properly constructed expression vector will generally comprise: at least one selectable marker expressible in animal cells for inserting the expression cassette of the recombinant product gene under the control of an upstream promoter region at a limited number of useful restriction sites. If used only for transient/episomal expression, the expression vector may also comprise an origin of replication, e.g.the origin of replication of the Eppstein Barr Virus (EBV) or SV40 virus, for self-replication/episomal maintenance in a eukaryotic host cell, but may be devoid of a selectable marker. Expression vectors are for example but not limited to linear DNA fragments, DNA fragments comprising a nuclear targeting sequence or optimized for interaction with transfection agents, animal viruses or suitable plasmids that can be shuttled and replicated between bacteria. Various common selection markers may be used, such as thymidine kinase (tk), dihydrofolate reductase (DHFR) or Glutamine Synthetase (GS). One preferred embodiment employs an expressible GS selection marker (Bebbington et al, 1992, "high level expression of recombinant antibodies in myeloma cells with glutamine synthetase gene as an amplifiable selection marker", Bio/Technology 10: 169-. The GS system is one of the only two systems that are particularly important for the production of therapeutic proteins. The GS system has a great time advantage over the dihydrofolate reductase (DHFR) system in the growth process, since it generally forms a highly productive cell line from the original transfectant, thus eliminating the need for gene amplification through multiple rounds of selection with increasing concentrations of selection reagents (Btown et al, 1992, "development of methods for producing recombinant antibodies using the Glutamine Synthetase (GS) system", Cytotechnology 9: 231-. Needless to say, equivalent to using the second transcription unit of the expression marker gene is: an expression unit can simultaneously express a product gene and a marker gene using a single cistron expression cassette, using, for example, commonly used internal ribosome entry sites. In contrast, the hot-spot IgG 2A sequences of the invention and the expression cassettes comprising the promoter and/or marker cassette do not necessarily have to be run in cis on the same expression vector; the elements may be carried by different vectors or DNA fragments which are co-transfected and then integrated at a unique tandem integration site on the chromosome.

Another object of the invention is to provide CHO host cells transfected with the DNA sequences of the invention. Other objects also include methods of transfecting such host cells and methods of expressing recombinant product genes in such host cells. The explanations and references in the description of the preferred embodiments of the present invention are equally applicable to the other objects of the present invention described above. It is to be noted that a host cell transfected with a DNA sequence or vector of the invention is understood to be a transiently transfected or stably transfected cell line. The present invention can employ various transfection techniques depending on the type of host cell, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc., which are well known in the art.

Suitable host cell lines may be various Chinese Hamster Ovary (CHO) cell lines (Puck et al, 1958, test medicine impurities 108: 945-. "host cell" refers to a cell that is capable of producing and expressing a desired recombinant protein product in culture. Suitable cell lines may be, for example, the CHO KI (ATCC CCL-61), CHOpro-3, CHO DG44, CHO P12 or the dhfr-CHO cell line DUK-BII (Chassin et al, PNAS77, 1980, 4216-4220) or DUXB11(Simonsen et al, PNAS80, 1983, 2495-2499). In CHO cells, the immunoglobulin gene locus is inactive, and thus chromatin is in a tightly packed (packeged) or closed state. Thus, due to this particular state of chromatin, any genetic construct integrated into an immunoglobulin locus cannot express a high level of recombinant protein unless the construct itself carries flanking locus regulatory regions that open up the expression cassette in chromatin on both sides. Furthermore, immunoglobulin gene sequences, particularly portions of their content, exhibit considerable interspecies differences, for example between mice and hamsters. Promoter or enhancer elements of immunoglobulin loci are both species-specific and tissue-specific and are active only in B cells. Various native immunoglobulin promoters may produce full-length transcripts encoding the entire IgG heavy chain, and the murine IgG 2A sequences of the present invention are capable of enhancing gene expression in CHO cells even in the absence of such native promoters. Preferably, the IgG 2A sequences of the invention do not have such a promoter. Surprisingly, the murine IgG 2A targeting sequence even enhanced gene expression in CHO cells after transient transfection of CHO cells with the expression vectors of the invention (fig. 1); such transient expression is a further preferred embodiment of the method of the invention. Typically in transient expression assays that occur 20-50 hours after transfection, the transfection vector remains an extrachromosomal element and does not integrate into the genome.

Media and culture methods suitable for mammalian cell lines are well known in the art and can be found, for example, in US 563162. Standard cell culture media for laboratory conical flask cultures or low-density cell cultures, modified according to the requirements of different cell types, are for example: roswell Park Memori Institute (RPMI)1640 medium (Morre, G., journal of the American medical Association, 199, p.519 f.1967), L-15 medium (Leibovitz, A. et al, journal of the American health, 78, 1p.173 ff.1963), Dulbecco's Modified Eagle's Medium (DMEM), Eagle's essential medium (MEM), Ham's F12 medium (Ham, R. et al, Proc. Natl.Acad.Sci.USA 53, p288 ff.1965) or Iscoves's modified DMEM without albumin, transferrin, and lecithin (Iscoves et al, journal of Experimental medicine 1, p.923 ff., 1978). For example, Hams's F10 or F12 media were specifically designed for CHO cell culture. Other media suitable for CHO cells can be found in EP-481791. It is known that such media can be supplemented with fetal bovine serum (FBS, also known as FCS) to provide an abundant natural source of hormones and growth factors. Animal cell culture is now a routine procedure, well described in scientific textbooks and manuals, e.g., r.ian fresnel, handbook of animal cell culture, 4 th edition (Wiley-loss/n.y., 2000).

Preferably, the cell culture medium of the present invention does not contain fetal bovine serum (FBS or FCS), and may be referred to as "serum-free". In serum-free media, cells typically require insulin and transferrin for optimal growth conditions. At least part of the transferrin may be replaced by a non-peptide chelator or an siderophore such as tropolone as described in WO94/02592, or by increasing the level of some organic iron, preferably with an antioxidant such as vitamin C. Most cell lines require one or more synthetic growth factors (including recombinant polypeptides), including, for example, Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), insulin-like growth factors I and II (IGFI, IGFII), and the like. Other factors that may be required include: prostaglandins, transport and binding proteins (e.g., ceruloplasmin, high and low density apolipoproteins, Bovine Serum Albumin (BSA)), hormones, including steroid hormones, and fatty acids. Preferably, the polypeptide factor assay is performed in steps in which the novel polypeptide factors are tested in the presence of known growth-promoting polypeptides. Those growth factors are synthetic or recombinant. There are various methods for animal culture, one of which is described below. First, conditions are determined under which cells survive and/or grow slowly for 3-6 days after transfer from serum-supplemented media. For most cell types, this depends at least in part on seeding density. Once the optimal hormone/growth factor/polypeptide supplement is found, the density of vaccination required for survival can be reduced. In a preferred embodiment, the cell culture medium is protein-free, i.e., free of fetal calf serum and various protein growth factor supplements or other proteins such as recombinant transferrin.

One possible embodiment of the method of the invention, i.e.recombinant protein expression and harvesting, is the high density growth of animal host cells in, for example, an industrial batch bioreactor. Then can proceedConventional downstream processing. For this purpose, a high-density growth medium must be used. Such high-density growth media are usually supplemented with nutrients such as full amino acids, energy sources such as glucose in the aforementioned amounts, inorganic salts, vitamins, trace elements (i.e. inorganic compounds in final concentration in micromolar), buffers, antioxidants such as the four nucleosides or corresponding nucleotides, (reduced) glutathione, vitamin C, etc., and other important components such as important membrane lipids, e.g. cholesterol or phosphatidylcholine, or lipid precursors, e.g. choline or inositol. Most or all of the above compounds in high density media are enriched (fortified) so that their content is higher than in the aforementioned standard media, which is seen in comparison of GB2251249 to RPMI1640, but the inorganic salts that are the basis for the regulation of osmotic pressure are not enriched. Preferably, the high density medium of the present invention is balanced and enriched for all amino acids except tryptophan in an amount exceeding 75mg per liter of medium. Preferably, the glutamine and/or asparagine content is more than 1g/L, more preferably more than 2g/L, in addition to achieving the overall amino acid requirement. In the context of the present invention, high density cell culture means that the population of animal cells has an instantaneous density of viable cells of at least 10⁵One/ml, at least 10⁶Preferably, the population is formed by the sequential assembly of single cells or low viable cell density inoculum in a constant or increasing amount of medium.

In a further preferred embodiment, the batch culture is a culture system wherein, in addition to controlling the glucose concentration by fed-batch, the cell culture is supplemented with at least glutamine, or with one or several other amino acids, preferably glycine, to maintain their concentration in the culture medium, as described in GB 2251249. More preferably, the addition of glutamine and optionally one or more other amino acids is combined with the addition of one or more energy sources, such as glucose, as described in EP-229809A. The first feeding is generally 25-60 hours after the start of the culture; for example, cell densities of up to 10⁶The addition was started at around one ml. It is well known that "glutaminolysis" in cultured animal cells (McKeehan et al, 1984, "in animal cells)Glutamine decomposition "in cultured cells, edited by m.j. morgan, Plenum press, new york, pp.11-150) may become an important energy source in the growth phase. The total amount of the additional glutamine and/or asparagine (for the substitution of glutamine by asparagine see Kurano, N et al, 1990, J. Biotechnology 15, 113-128) is generally 0.5-10g/L, preferably 1-2g/L, of culture; the total amount of other amino acids that may be included in the feed may be between 10 and 300mg/L culture, in particular the amount of glycine, lysine, arginine, valine, isoleucine and leucine may be relatively high, at least 150-200 mg. The feed may be a shot-addition or a continuous pumped addition, preferably an almost continuous pumped feed to the bioreactor. Needless to say, during batch culture, the pH in the bioreactor is carefully controlled to the physiological pH most suitable for a given cell line by the addition of alkali or buffer. When glucose is used as an energy source, the total glucose feed is generally 1-10g/L, preferably 3-6g/L, of culture. The feed preferably contains, in addition to amino acids, small amounts of, for example, 5 to 20mg/L of culture choline. More preferably, the choline supplementation is combined with the ethanolamine supplementation, as described in US6048728, especially with simultaneous glutamine supplementation. Needless to say, if the GS-tagged system is used, the glutamine requirement is lower than that of the non-GS-tagged system, and ammonia and poisoning are caused by accumulation of excess glutamine other than endogenously produced glutamine. For GS, glutamine in the medium or feed can generally be replaced with its equivalent and/or precursor, i.e., asparagine and/or glutamic acid.

It is a further object of the present invention to design an expression vector comprising at least one (first) product gene transcription unit which produces a protein upon expression in a host cell and which is regulated by the mouse cytomegalovirus promoter (mCMV promoter), and a second transcription unit which comprises a Glutamine Synthetase (GS) marker gene. The product gene, or so-called gene of interest (GOI), may be, for example, an immunoglobulin coding sequence. The glutamine synthetase marker gene is a sequence encoding GS having an enzymatic activity, including a natural sequence or a variant thereof. The foregoing definitions of "functional variants" including preferred ranges of sequence homology apply equally here. Preferably, the GS marker gene is a mammalian GS marker gene or a derivative thereof. Surprisingly, the transfection efficiency of such vectors into CHO cells was much higher compared to expression vectors in which the gene of interest in the first transcription unit was regulated by the hCMV promoter. This is not because the transcriptional activity of the mCMV promoter in CHO cells is much higher than that of the mCMV promoter; it is generally believed that higher metabolic loads reduce the survival of clones after transfection, resulting in fewer transfectants. Thus, this effect cannot be simply correlated with the amount of protein product expressed or unexpected toxicity that is detrimental to the growth of the transfectants. Indeed, the above findings are largely unexpected to those skilled in the art.

Other objects of the invention include animal host cells, particularly CHO cells, transfected with expression vectors that can remain extrachromosomal and that can also stably integrate into the genome, and related transfection methods. Similarly, animal cells, in particular CHO cells, are transfected with two or more gene fragments which form in vivo functional equivalents of the transcription unit which is the object of the invention, which are also included in the definition of transfected host cells. Preferably, the host cell is a stably transfected cell, i.e.both the first and second transcription units are integrated into the chromosome.

Another object of the present invention is to provide the use of the mCMV promoter for increasing transfection efficiency in CHO cells, preferably transfection with the following expression vectors: the expression vector comprises at least one first product gene transcription unit which produces a protein upon expression in a host cell and which is regulated by the mouse cytomegalovirus promoter (mCMV promoter), and a second transcription unit which comprises a Glutamine Synthetase (GS) marker gene. The first and second expression sequences are carried by different vectors or as gene segments in different expression units. Furthermore, a recombinant CHO cell expressing GS can be transfected with the first transcription unit with mCMV. According to the present invention, "increasing transfection efficiency" is achieved by replacing the transfection efficiency in the presence of the mCMV promoter and the expression vector of the invention with the transfection efficiency in the same expression vector of mCMV in the same host cell under the same transfection conditions and the same cell culture conditions with the nucleic acid sequence described in US5658759 and of the invention of seq id NO: 3, by comparison with the transfection efficiency of the hCMV-first intron enhancer/promoter construct. For a given identical product gene, the hCMV-intron MIE-promoter construct is the reference standard for determining the transfection efficiency enhancing effect. Preferably, mCMV increases transfection efficiency by at least 10-fold.

The above definitions apply equally to the various objects of the invention. It is emphasized that the murine IgG 2A targeting sequence is not a prerequisite for achieving the objects of the invention.

Murine cytomegalovirus (mCMV) is a member of the highly diverse herpesviridae family. Even macrophage viruses of different hosts may differ significantly. For example, mCMV differs considerably from human macrophage virus (hCMV) in biological properties, Immediate Early (IE) gene organization, and overall nucleic acid sequence. Furthermore, the 235kbp mCMV has no internal and terminal repeats characteristic of hCMV. Therefore, the mCMV genome has no isoforms (Ebeling, A et al, 1983, J. Virol 47, 421- & 433: Mercer J. A. et al, (1983), virology 129, 94-126). According to the invention, a promoter region or a functional fragment thereof which corresponds essentially to the approximately 2.1kb PstI fragment described in US4968615 can be used. In a more preferred embodiment, the cMCV promoter fragment used comprises the transcription start (+0) to this pre-500 bp sequence. Surprisingly, this fragment driven expression more strongly than the promoter cassette which was 800bp further forward from-500 bp. In the best embodiment, the promoter core region, i.e., the Xho I restriction site from the transcription start site to about-150 bp upstream, or even from the natural transcription start site to-100 bp upstream, is used. Needless to say, the transcription initiation point may be artificially modified in order to contain an appropriate restriction site for insertion of the recombinant product gene.

According to the invention, the first transcription unit regulated by the mCMV promoter may comprise at least one intron sequence. This is common knowledge in the art, in order to stabilize RNA transcripts to increase the efficiency of protein synthesis from the corresponding mRNA. However, for efficiency of protein synthesis, the inclusion of the natural intron of mCVM within the mCMV promoter construct is not suggested, regardless of the effect on transfection efficiency. In contrast to the case of the hCMV promoter (US5591639), such a first intron of mCMV reduces the expression of recombinant genes under the mCMV promoter and is therefore not a preferred embodiment.

The sequence listing gives a preferred, possible embodiment of the GS marker gene cassette. SEQ ID NO: 1(pee15.1hcmv/GFP + hot spot) and SEQ ID NO: 2(pEE14.4hCMV/GFP) is an example of a suitable SV40 (early, late) promoter regulated GS gene cassette, which is a moderately weak promoter, and which also contains a GFP (Green fluorescent protein) expression cassette under the control of the hCMV promoter. SEQ ID NO: 1 is the GS cDNA sequence under the control of the SV40 early promoter, the details of which are shown in FIG. 3. SEQ ID NO: 2a synthetic GS minigene cassette comprising an intron under the control of the SV40 late promoter. CHO cells are not naturally glutamine auxotrophic and so the selection protocol described by Cockett et al (1990, "tissue inhibitors that use glutamine synthetase gene amplification to highly express metalloproteinases in Chinese Hamster Ovary (CHO) cells", Bio/Technology 8: 662-667) can be performed. This reference also exemplifies transfection methods suitable for CHO cells: for example, classical calcium phosphate precipitation or newer lipofection techniques can be used. Transfection efficiency is generally defined as the number of transfection-positive cells (transient transfection) or clones (stable transfection after a selection period) in the transfected cell population. The effect of the invention can be achieved, for example, by lipofection (any commercially available reagents and manufacturer's instructions) with a nucleic acid comprising SEQ ID NO: 3(pEE12.4hCMV-GFP + SV40 early promoter/GS cDNA) or SEQ ID NO: 4(pEE 12.4mCMV-GFP + SV40 early promoter/GS cDNA) was transfected into CHO-K1 cells for expression. The transfected cells can be cultured in a variety of conventional media. These media may be the aforementioned serum-containing media or serum-free media. Preferably the serum-containing medium is supplemented, especially with at least 1% (v/v) fetal serum, most preferably at least 5% (v/v) fetal serum, such as fetal bovine serum. Another preferred embodiment employs electroporation transfection.

Examples

Experiment 1

Transient expression and stable expression of GFP vector containing hotspot sequence in CHO-K1

CHO-K1 cells (ATCC CCL-6) were adapted and cultured in normal cell culture medium (GMEM-S) containing 10% FCS. For GS selection, the medium must be completely glutamine free, as shown in table 1 below: therefore, dialyzed FCS must be used. The culture was carried out at 36.5 ℃ and 125rpm in a shaking flask. Green fluorescence of the transfectant cell population was measured in FACS using lipofection (Superfectin TM, Gibco, UK) with an excitation wavelength of 488 nm. For each GS/GFP vector construct, 5 transfections were performed, and all data are averages from analysis of 5 independent cell populations. Starting from transient transfectants 48 hours after transfection, the mean fluorescence was determined by picking the number of cells vs. the highest 10% of the live cells in the fluorescence map (FIG. 1). The live cell population was picked by gating (gating) in a Forward vs. side scatter plot.

To generate stable transfectants, GS markers were selected 24 hours post-transfection by adding 25 μ MMSX (methionine sulphoximine, Crockert et al, ibd.) to glutamine-free medium, continuing the culture and periodically dividing the culture for 26 days. The effect of the median values of other amino acid levels on the MSX selection intensity was observed, see Bebbington et al, US 5827739. Fluorescence analysis was then performed as described above (FIG. 2) with non-transfected cells as negative controls. SEQ ID NO: 1 shows a hot spot vector (pEE15.1' hCMV + hot spot) driving expression of GFP under the control of the hCMV promoter containing the entire first intron of CMV, which is actually the insertion of the GFP sequence into the Eco RI restriction site of the polylinker region within the pEE15.1 vector shown in FIG. 3. Correspond toIn SEQ ID NO: the pEE12.4 'hCMV of 3 differs from the pEE 15.1' hCMV + hotspot only in that it does not contain a 5.1kb BamH 1 fragment containing IgG 2A sequences. pEE12.4 was used as a vector control. Another vector control is pEE12.4 'hCMV (Kozak-)' which is a Kozak sequence of a cloning site that will coincide with the translational start by primer-directed mutagenesis (Sambrook et al, molecular cloning, Cold spring harbor, 1983) (GCCGCCA CCA)CCATGG) Frameshift mutations to functional Kozak sequences (ACC)ATGGT CCATGG) Thereby eliminating the original Kozak translation initiation point. SEQ ID NO: the vector shown in 1 was further deleted for the 400bp regulator region of the hCMV enhancer portion and for the enhancer element at-750 bp upstream of the transcription start point, thereby forming pEE15.1 'hCMV (mod-)/GS cDNA'. As shown in fig. 2, the GS cDNA cassette of pEE15.1 (fig. 3) was exchanged for SEQ ID NO: 2, pEE14.4 'hCMV (mod-)/GS (minigene) of GFP, to form pEE 15.1' hCMV (mod-)/GS minigene. Thus, all transfected cells contained a plasmid vector containing the GFP coding sequence. The GS minigene contains the first intron of a single GS gene and a segment of about 1kb of 3' flanking DNA, under the control of the SV40 late promoter; it is believed that the 3' portion of the genomic GS DNA increases the copy number of the vector DNA and thus the GS content in the transfected cells (see US4770359, Bebbington et al). Although the hCMV vectors used in the present invention all expressed the GS marker gene in the GS cDNA sequence, the GS minigene was used as a further control to exclude the effects that GS copy number and expression level may have.

To analyze the production and expression of stably transfected GHO cells, transfection was performed with the linearized hot spot vector pEE15.1 'hCMV + hot spot' vector. The Sal I linearized plasmid was cut in the IgG 2A containing sequence portion, and the ends of the free DNA might promote recombination in the genomic region flanked by DNA with a certain degree of homology to the ends, so that the possible targeting effect of murine IgG 2A in CHO cells could be tested. The Pvu I cuts into the bacterial lactamase marker gene and thus promotes heterologous random recombination. Indeed, the higher fluorescence in the Pvu I linearized transfectants indicates that vector linearization has some effect, and the targeting of CHO cells to immunoglobulin loci does not explain the efficacy of the invention. Furthermore, the increased promoter activity also observed in transient transfected cell populations correlates closely with the relative strength of the individual vector constructs. Apparently, no genomic integration occurred during the early phase of transfection.

FIG. 3 shows the vector pEE15.1 of about 12830 bp. For a detailed description of the GS marker and the hCMV-p/intron expression cassette see US5827739 and US 5591639. pEE15.1 is a possible embodiment of the expression vector of the present invention, but does not have a DNA sequence encoding a recombinant product protein inserted into the polylinker site. SEQ ID NO: 1 shows the sequence of the entire I3535bp of the GFP-containing pEE15.1 construct: wherein, the GFP coding sequence is inserted into an Eco RI restriction site in an reading frame, and the site is positioned at 12814 bases in the middle; this unique restriction site contains the ATG start codon and optimizes the Kozak sequence context of the start codon, as detailed in US 5591639. Thus, GFP protein expression is under the control of the hCMV-important immediate early gene promoter (hCMV-MIE or abbreviated hCMV), followed immediately by the first intron of the hCMV-MIE gene, followed by the Nco I site (see US 5591639). The SV40 polyA site downstream of the polylinker region ensures the completion of polyadenylation. pEE15.1 also contains a cDNA sequence encoding hamster Glutamine Synthetase (GS) under the control of the SV40 early promoter, followed by the SV40 intron + poly A sequence. The IgG 2A locus or "hot spot" sequences (shaded boxes CH1, Hi, CH2, CH3, M1 and M2 represent the heavy chain constant region, hinge region, cell membrane anchoring region) are the 5.1kb BamH I fragment of the murine IgG 2A locus described in WO9517516 and its references. Unique Pvu I and Sal I restriction sites are shown.

Experiment 2

Transfection of CHO cells with mCMV p12.4-GFP construct (SEQ ID NO: 4) by electroporation

Coanda CHO-K1 cells (ATCC CCL-61) were cultured in Iscoves's DMEM medium containing 2mM glutamine supplemented with 10% FCS as described in EP-48791.Alternatively, G-MEM medium containing 2mM glutamine as shown in Table 1 can be used before GS marker selection as described in experiment 1. Cells were detached, pelleted, then resuspended twice in serum-free medium to 5.3X 10/ml⁶Density of individual cells. 750. mu.l of each batch, 4X 10 in total⁶Individual cells were subjected to electroporation. For example, methods in molecular biology, ed.J., JA Nickoloff, Humana Press, 1995, Vol.48/Chap.8: electroporation of animal cells and electrofusion method the electroporation is carried out as described in the section "animal cell electroporation and electrofusion method". The p12.4mCMV-GFP vector DNA (SEQ ID NO: 4) was linearized. Mu.l (20. mu.g) of DNA was added to 750. mu.l of cells in an electroporation cuvette and electroporation was carried out at 300V/750. mu.FD for about 12-14 milliseconds. Then, 800. mu.l of the cells were transferred to 25ml modified Glasgow-MEM (GMEM, Gibco) medium (containing 10% fetal serum but no glutamine, see Table 1 for details) in T75 Erlenmeyer flasks for GS selection. 12.9ml were divided into a second T75 conical flask, 37 ℃ and 10% CO₂The next day of overnight medium culture, GS-selective GMEM medium supplemented with 10% FBS + 33.3. mu.M MSX was added. Thus, the final concentration of MSX was about 25. mu.M. After an additional 26 days of incubation, the number of colonies per flask, i.e., transfectants, was counted. Microscopic examination of each flask was performed using a standard inverted microscope, and positive clones emitted bright green light, thus allowing easy spotting.

SEQ ID NO: 4 compared to the colony produced by the mCMV construct shown in SEQ ID NO: 3 hCMV construct transfected cells 20-fold more. The two vector constructs differed only in that the CMV promoter driving GFP expression differed, and the rest of the vector was identical (including the GS marker; cDNA GS-marker cassette for p 12.4). If cells were diluted immediately after transfection and added to 96 plates, mCMV cells still formed more colonies (> 400) than hCMV transfected cells (about 45).

Table 1: GS selection Medium

A. Stock solution

1. Autoclaving with double distilled water (400 ml each)

2.10 XGlasgow MEM (GMEM) medium, without glutamine (GIBCO: 042-2541, in UK). Storing at 4 ℃.

3.7.5% sodium bicarbonate (GIBCO: 043-05080 in UK; 670-5080 in US). Storing at 4 ℃.

4.100 × non-essential amino acids (NEAA) (GIBCO: 043-01140, in UK; 320-1140, in US). Storing at 4 ℃.

5.100 Xglutamic acid + asparagine (G + A): 600mg of glutamic acid and 600mg of asparagine (Sigma) were added. 100ml of the mixture was prepared with distilled water and sterilized by filtration through a 2 μm filter (Nalgene). Storing at 4 ℃.

6.100mM sodium pyruvate (GIBCO: 043-01360 in UK; 320-1360 in US)

7.50 × nucleosides: 35mg adenosine

35mg of guanosine

35mg Cytidine

35mg uridine

12mg of thymidine

All purchased from Sigma. Preparing 100ml solution with water, filtering, sterilizing, and storing at-20 deg.C in 10ml portions.

8. Dialyzed FCS (GIBCO: 014- & 06300). Heat-inactivated at 56 ℃ for 30 minutes and stored at-20 ℃. For GS selection, dialyzed FCS must be used.

9. 5000 units per ml of penicillin-streptomycin (P/S: GIBCO: 043-05070 in UK; 600-5070 in US).

10.100mM L.MSX (Sigma): 18mg/ml PBS solution was prepared. Filtering for sterilization, and storing at-20 deg.C.

B. Preparation of the culture Medium

The following substances are sequentially added according to the following sequence by aseptic technique to prepare GMEM-S culture medium.

1. 400ml of water

2.10×GMEM 50ml

3. Sodium bicarbonate 18.1ml

4.NEAA 5ml

5.G+A 5ml

6. Sodium pyruvate 5ml

7. Nucleoside 10ml

8. Dialyzed FCS 50ml

9. Penicillin-streptomycin 5ml

GMEM-S medium contains nonessential amino acids, alanine, aspartic acid, glycine, proline and serine (100. mu.M), glutamic acid and asparagine (500. mu.M), adenosine, guanosine, cytidine and uridine (30. mu.M), and thymidine (10. mu.M).

Sequence listing

<110> Longsha biomedical products of England Ltd

<120> method for expressing recombinant protein in CHO cell

<130>049369

<150>GB0216648.6

<151>2002-07-19

<160>4

<170>PatentIn version 3.1

<210>1

<211>6679

<212>DNA

<213> Hamster (Hamster sp.)

<220>

<221>misc_feature

<223> seq. id.no. 4: circular plasmid GS vector p12.4 short mCMV-GFP/clone 3

<400>1

gaattcattg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 60

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 120

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 180

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 240

tgtctggcgg ccgcgacctg caggcgcaga actggtaggt atggaagatc cctcgagatc 300

cattgtgctg gcggtaggcg agcagcgcct gcctgaagct gcgggcattc ccagtcagaa 360

atgagcgcca gtcgtcgtcg gctctcggca ccgaagtgct atgattctcc gccagcatgg 420

cttcggccag tgcgtcgagc agcgcccgct tgttcctgaa gtgccagtaa agcgccggct 480

gctgaacccc caaccgttcc gccagtttgc gtgtcgtcag accgtctacg ccgacctcgt 540

tcaacaggtc cagggcggca cggatcactg tattcggctg caactttgtc atgcttgaca 600

ctttatcact gataaacata atatgtccac caacttatca gtgataaaga atccgcgcca 660

gcacaatgga tctcgaggtc gagggatctc tagaggatcc atattcgcgg gcatcaccgg 720

cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc 780

tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc 840

cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa 900

cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg 960

acctcgggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1020

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1080

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1140

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1200

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 1260

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 1320

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 1380

tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 1440

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 1500

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1560

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 1620

aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 1680

tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 1740

cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 1800

catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 1860

ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 1920

aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 1980

ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2040

caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2100

attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2160

agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 2220

actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 2280

ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 2340

ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 2400

gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 2460

atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 2520

cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 2580

gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 2640

gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 2700

ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 2760

gacattaacc tataaaaata ggcgtatcac gaggccctga tggctctttg cggcacccat 2820

cgttcgtaat gttccgtggc accgaggaca accctcaaga gaaaatgtaa tcacactggc 2880

tcaccttcgg gtgggccttt ctgcgtttat aaggagacac tttatgttta agaaggttgg 2940

taaattcctt gcggctttgg cagccaagct agatccggct gtggaatgtg tgtcagttag 3000

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 3060

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 3120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 3180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 3240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 3300

gcctaggctt ttgcaaaaag ctagcttggg gccaccgctc agagcacctt ccaccatggc 3360

cacctcagca agttcccact tgaacaaaaa catcaagcaa atgtacttgt gcctgcccca 3420

gggtgagaaa gtccaagcca tgtatatctg ggttgatggt actggagaag gactgcgctg 3480

caaaacccgc accctggact gtgagcccaa gtgtgtagaa gagttacctg agtggaattt 3540

tgatggctct agtacctttc agtctgaggg ctccaacagt gacatgtatc tcagccctgt 3600

tgccatgttt cgggacccct tccgcagaga tcccaacaag ctggtgttct gtgaagtttt 3660

caagtacaac cggaagcctg cagagaccaa tttaaggcac tcgtgtaaac ggataatgga 3720

catggtgagc aaccagcacc cctggtttgg aatggaacag gagtatactc tgatgggaac 3780

agatgggcac ccttttggtt ggccttccaa tggctttcct gggccccaag gtccgtatta 3840

ctgtggtgtg ggcgcagaca aagcctatgg cagggatatc gtggaggctc actaccgcgc 3900

ctgcttgtat gctggggtca agattacagg aacaaatgct gaggtcatgc ctgcccagtg 3960

ggaactccaa ataggaccct gtgaaggaat ccgcatggga gatcatctct gggtggcccg 4020

tttcatcttg catcgagtat gtgaagactt tggggtaata gcaacctttg accccaagcc 4080

cattcctggg aactggaatg gtgcaggctg ccataccaac tttagcacca aggccatgcg 4140

ggaggagaat ggtctgaagc acatcgagga ggccatcgag aaactaagca agcggcaccg 4200

gtaccacatt cgagcctacg atcccaaggg gggcctggac aatgcccgtg gtctgactgg 4260

gttccacgaa acgtccaaca tcaacgactt ttctgctggt gtcgccaatc gcagtgccag 4320

catccgcatt ccccggactg tcggccagga gaagaaaggt tactttgaag accgcggccc 4380

ctctgccaat tgtgacccct ttgcagtgac agaagccatc gtccgcacat gccttctcaa 4440

tgagactggc gacgagccct tccaatacaa aaactaatta gactttgagt gatcttgagc 4500

ctttcctagt tcatcccacc ccgccccaga gagatctttg tgaaggaacc ttacttctgt 4560

ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa 4620

tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca 4680

acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga aaacctgttt 4740

tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca acattctact 4800

cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga attgctaagt 4860

tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat ttacaccaca 4920

aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt aacctttata 4980

agtaggcata acagttataa tcataacata ctgttttttc ttactccaca caggcataga 5040

gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa 5100

ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa tcagccatac 5160

cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 5220

acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 5280

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 5340

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctctagct tcgtgtcaag 5400

gacggtgagg cgcgcctact gagtcattag ggactttcca atgggttttg cccagtacat 5460

aaggtcaata ggggtgaatc aacaggaaag tcccattgga gccaagtaca ctgagtcaat 5520

agggactttc cattgggttt tgcccagtac aaaaggtcaa tagggggtga gtcaatgggt 5580

ttttcccatt attggcacgt acataaggtc aataggggtg agtcattggg tttttccagc 5640

caatttaat taaaacgccat gtactttccc accattgacg tcaatgggct attgaaacta 5700

atgcaacgtg acctttaaac ggtactttcc catagctgat taatgggaaa gtaccgttct 5760

cgagccaata cacgtcaatg ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt 5820

ttcccctgga aattccatat tggcacgcat tctattggct gagctgcgtt ctacgtgggt 5880

ataagaggcg cgaccagcgt cggtaccgtc gcagtcttcg gtctgaccac cgtagaacgc 5940

agaagcttgc cgccaccatg gtgagcaagc agatcctgaa gaacaccggc ctgcaggaga 6000

tcatgagctt caaggtgaac ctggagggcg tggtgaacaa ccacgtgttc accatggagg 6060

gctgcggcaa gggcaacatc ctgttcggca accagctggt gcagatccgc gtgaccaagg 6120

gcgcccccct gcccttcgcc ttcgacatcc tgagccccgc cttccagtac ggcaaccgca 6180

ccttcaccaa gtaccccgag gacatcagcg acttcttcat ccagagcttc cccgccggct 6240

tcgtgtacga gcgcaccctg cgctacgagg acggcggcct ggtggagatc cgcagcgaca 6300

tcaacctgat cgaggagatg ttcgtgtacc gcgtggagta caagggccgc aacttcccca 6360

acgacggccc cgtgatgaag aagaccatca ccggcctgca gcccagcttc gaggtggtgt 6420

acatgaacga cggcgtgctg gtgggccagg tgatcctggt gtaccgcctg aacagcggca 6480

agttctacag ctgccacatg cgcaccctga tgaagagcaa gggcgtggtg aaggacttcc 6540

ccgagtacca cttcatccag caccgcctgg agaagaccta cgtggaggac ggcggcttcg 6600

tggagcagca cgagaccgcc atcgcccagc tgaccagcct gggcaagccc ctgggcagcc 6660

tgcacgagtg ggtgtaata 6679

<210>2

<211>8251

<212>DNA

<213> Hamster (Hamster sp.)

<220>

<221>misc_feature

<223> seq. id.no. 3: circular plasmid GS vector p12.4hCMVP-GFP/clone 13

<400>2

gaattcattg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 60

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 120

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 180

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 240

tgtctggcgg ccgcgacctg caggcgcaga actggtaggt atggaagatc cctcgagatc 300

cattgtgctg gcggtaggcg agcagcgcct gcctgaagct gcgggcattc ccagtcagaa 360

atgagcgcca gtcgtcgtcg gctctcggca ccgaagtgct atgattctcc gccagcatgg 420

cttcggccag tgcgtcgagc agcgcccgct tgttcctgaa gtgccagtaa agcgccggct 480

gctgaacccc caaccgttcc gccagtttgc gtgtcgtcag accgtctacg ccgacctcgt 540

tcaacaggtc cagggcggca cggatcactg tattcggctg caactttgtc atgcttgaca 600

ctttatcact gataaacata atatgtccac caacttatca gtgataaaga atccgcgcca 660

gcacaatgga tctcgaggtc gagggatctc tagaggatcc atattcgcgg gcatcaccgg 720

cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc 780

tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc 840

cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa 900

cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg 960

acctcgggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1020

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1080

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1140

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1200

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 1260

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 1320

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 1380

tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 1440

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 1500

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1560

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 1620

aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 1680

tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 1740

cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 1800

catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 1860

ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 1920

aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 1980

ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2040

caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2100

attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2160

agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 2220

actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 2280

ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 2340

ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 2400

gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 2460

atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 2520

cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 2580

gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 2640

gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 2700

ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 2760

gacattaacc tataaaaata ggcgtatcac gaggccctga tggctctttg cggcacccat 2820

cgttcgtaat gttccgtggc accgaggaca accctcaaga gaaaatgtaa tcacactggc 2880

tcaccttcgg gtgggccttt ctgcgtttat aaggagacac tttatgttta agaaggttgg 2940

taaattcctt gcggctttgg cagccaagct agatccggct gtggaatgtg tgtcagttag 3000

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 3060

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 3120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 3180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 3240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 3300

gcctaggctt ttgcaaaaag ctagcttggg gccaccgctc agagcacctt ccaccatggc 3360

cacctcagca agttcccact tgaacaaaaa catcaagcaa atgtacttgt gcctgcccca 3420

gggtgagaaa gtccaagcca tgtatatctg ggttgatggt actggagaag gactgcgctg 3480

caaaacccgc accctggact gtgagcccaa gtgtgtagaa gagttacctg agtggaattt 3540

tgatggctct agtacctttc agtctgaggg ctccaacagt gacatgtatc tcagccctgt 3600

tgccatgttt cgggacccct tccgcagaga tcccaacaag ctggtgttct gtgaagtttt 3660

caagtacaac cggaagcctg cagagaccaa tttaaggcac tcgtgtaaac ggataatgga 3720

catggtgagc aaccagcacc cctggtttgg aatggaacag gagtatactc tgatgggaac 3780

agatgggcac ccttttggtt ggccttccaa tggctttcct gggccccaag gtccgtatta 3840

ctgtggtgtg ggcgcagaca aagcctatgg cagggatatc gtggaggctc actaccgcgc 3900

ctgcttgtat gctggggtca agattacagg aacaaatgct gaggtcatgc ctgcccagtg 3960

ggaactccaa ataggaccct gtgaaggaat ccgcatggga gatcatctct gggtggcccg 4020

tttcatcttg catcgagtat gtgaagactt tggggtaata gcaacctttg accccaagcc 4080

cattcctggg aactggaatg gtgcaggctg ccataccaac tttagcacca aggccatgcg 4140

ggaggagaat ggtctgaagc acatcgagga ggccatcgag aaactaagca agcggcaccg 4200

gtaccacatt cgagcctacg atcccaaggg gggcctggac aatgcccgtg gtctgactgg 4260

gttccacgaa acgtccaaca tcaacgactt ttctgctggt gtcgccaatc gcagtgccag 4320

catccgcatt ccccggactg tcggccagga gaagaaaggt tactttgaag accgcggccc 4380

ctctgccaat tgtgacccct ttgcagtgac agaagccatc gtccgcacat gccttctcaa 4440

tgagactggc gacgagccct tccaatacaa aaactaatta gactttgagt gatcttgagc 4500

ctttcctagt tcatcccacc ccgccccaga gagatctttg tgaaggaacc ttacttctgt 4560

ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa 4620

tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca 4680

acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga aaacctgttt 4740

tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca acattctact 4800

cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga attgctaagt 4860

tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat ttacaccaca 4920

aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt aacctttata 4980

agtaggcata acagttataa tcataacata ctgttttttc ttactccaca caggcataga 5040

gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa 5100

ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa tcagccatac 5160

cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 5220

acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 5280

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 5340

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctagcttc gtgtcaagga 5400

cggtgactgc agtgaataat aaaatgtgtg tttgtccgaa atacgcgttt tgagatttct 5460

gtcgccgact aaattcatgt cgcgcgatag tggtgtttat cgccgataga gatggcgata 5520

ttggaaaaat cgatatttga aaatatggca tattgaaaat gtcgccgatg tgagtttctg 5580

tgtaactgat atcgccattt ttccaaaagt gatttttggg catacgcgat atctggcgat 5640

agcgcttata tcgtttacgg gggatggcga tagacgactt tggtgacttg ggcgattctg 5700

tgtgtcgcaa atatcgcagt ttcgatatag gtgacagacg atatgaggct atatcgccga 5760

tagaggcgac atcaagctgg cacatggcca atgcatatcg atctatacat tgaatcaata 5820

ttggccatta gccatattat tcattggtta tatagcataa atcaatattg gctattggcc 5880

attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt 5940

accgccatgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 6000

agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 6060

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 6120

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 6180

ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 6240

atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 6300

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 6360

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 6420

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 6480

attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 6540

agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 6600

ccgggaccga tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc 6660

caagagtgac gtaagtaccg cctatagagt ctataggccc acccccttgg cttcttatgc 6720

atgctatact gtttttggct tggggtctat acacccccgc ttcctcatgt tataggtgat 6780

ggtatagctt agcctatagg tgtgggttat tgaccattat tgaccactcc cctattggtg 6840

acgatacttt ccattactaa tccataacat ggctctttgc cacaactctc tttattggct 6900

atatgccaat acactgtcct tcagagactg acacggactc tgtattttta caggatgggg 6960

tctcatttat tatttacaaa ttcacatata caacaccacc gtccccagtg cccgcagttt 7020

ttattaaaca taacgtggga tctccacgcg aatctcgggt acgtgttccg gacatgggct 7080

cttctccggt agcggcggag cttctacatc cgagccctgc tcccatgcct ccagcgactc 7140

atggtcgctc ggcagctcct tgctcctaac agtggaggcc agacttaggc acagcacgat 7200

gcccaccacc accagtgtgc cgcacaaggc cgtggcggta gggtatgtgt ctgaaaatga 7260

gctcggggag cgggcttgca ccgctgacgc atttggaaga cttaaggcag cggcagaaga 7320

agatgcaggc agctgagttg ttgtgttctg ataagagtca gaggtaactc ccgttgcggt 7380

gctgttaacg gtggagggca gtgtagtctg agcagtactc gttgctgccg cgcgcgccac 7440

cagacataat agctgacaga ctaacagact gttcctttcc atgggtcttt tctgcagtca 7500

ccgtccttga cacgaagctt gccgccacca tggtgagcaa gcagatcctg aagaacaccg 7560

gcctgcagga gatcatgagc ttcaaggtga acctggaggg cgtggtgaac aaccacgtgt 7620

tcaccatgga gggctgcggc aagggcaaca tcctgttcgg caaccagctg gtgcagatcc 7680

gcgtgaccaa gggcgccccc ctgcccttcg ccttcgacat cctgagcccc gccttccagt 7740

acggcaaccg caccttcacc aagtaccccg aggacatcag cgacttcttc atccagagct 7800

tccccgccgg cttcgtgtac gagcgcaccc tgcgctacga ggacggcggc ctggtggaga 7860

tccgcagcga catcaacctg atcgaggaga tgttcgtgta ccgcgtggag tacaagggcc 7920

gcaacttccc caacgacggc cccgtgatga agaagaccat caccggcctg cagcccagct 7980

tcgaggtggt gtacatgaac gacggcgtgc tggtgggcca ggtgatcctg gtgtaccgcc 8040

tgaacagcgg caagttctac agctgccaca tgcgcaccct gatgaagagc aagggcgtgg 8100

tgaaggactt ccccgagtac cacttcatcc agcaccgcct ggagaagacc tacgtggagg 8160

acggcggctt cgtggagcag cacgagaccg ccatcgccca gctgaccagc ctgggcaagc 8220

ccctgggcag cctgcacgag tgggtgtaat a 8251

<210>3

<211>10369

<212>DNA

<213> Hamster (Hamster sp.)

<220>

<221>misc_feature

<223> seq. id.no. 2: circular plasmid GS-minigene vector p14.4Delta regulon (mod-) hCMVP-GFP/clone 6

<400>3

gaattcattg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 60

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 120

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 180

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 240

tgtctggcgg ccgcgacctg caggcgcaga actggtaggt atggaagatc cctcgagatc 300

cattgtgctg gcggtaggcg agcagcgcct gcctgaagct gcgggcattc ccagtcagaa 360

atgagcgcca gtcgtcgtcg gctctcggca ccgaagtgct atgattctcc gccagcatgg 420

cttcggccag tgcgtcgagc agcgcccgct tgttcctgaa gtgccagtaa agcgccggct 480

gctgaacccc caaccgttcc gccagtttgc gtgtcgtcag accgtctacg ccgacctcgt 540

tcaacaggtc cagggcggca cggatcactg tattcggctg caactttgtc atgcttgaca 600

ctttatcact gataaacata atatgtccac caacttatca gtgataaaga atccgcgcca 660

gcacaatgga tctcgaggtc gagggatctc tagaggatcc atattcgcgg gcatcaccgg 720

cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc 780

tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc 840

cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa 900

cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg 960

acctcgggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1020

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1080

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1140

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1200

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 1260

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 1320

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 1380

tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 1440

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 1500

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1560

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 1620

aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 1680

tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 1740

cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 1800

catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 1860

ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 1920

aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 1980

ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2040

caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2100

attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2160

agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 2220

actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 2280

ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 2340

ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 2400

gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 2460

atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 2520

cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 2580

gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 2640

gggttattgt ctcatgagcg gatacatatt-tgaatgtatt tagaaaaata aacaaatagg 2700

ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 2760

gacattaacc tataaaaata ggcgtatcac gaggccctga tggctctttg cggcacccat 2820

cgttcgtaat gttccgtggc accgaggaca accctcaaga gaaaatgtaa tcacactggc 2880

tcaccttcgg gtgggccttt ctgcgtttat aaggagacac tttatgttta agaaggttgg 2940

taaattcctt gcggctttgg cagccaagct agatccagct ttttgcaaaa gcctaggcct 3000

ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct 3060

gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt 3120

aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg 3180

ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa 3240

ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct 3300

aactgacaca cattccacag ggaagctagc ttggaattaa ttccccgccc ccttccaata 3360

caaaaactaa ttagactttg agtgatcttg agcctttcct agtttttgta ttggaagggc 3420

tcgtcgccag tctcattgag aaggcatgtg cggacgatgg cttctgtcac tgcaaagggg 3480

tcacaattgg cagaggggcg gcggtcttca aagtaacctt tcttctcctg ccgagccgag 3540

aatgggagta gagccgactg cttgattccc acaccaatct cctcgccgct ctcacttcgc 3600

ctcgttctcg tggctcgtgg ccctgtccac cccgtccatc atcccgccgg ccaccgctca 3660

gagcaccttc caccatggcc acctcagcaa gttcccactt gaacaaaaac atcaagcaaa 3720

tgtacttgtg cctgccccag ggtgagaaag tccaagccat gtatatctgg gttgatggta 3780

ctggagaagg actgcgctgc aaaacccgca ccctggactg tgagcccaag tgtgtagaag 3840

agttacctga gtggaatttt gatggctcta gtacctttca gtctgagggc tccaacagtg 3900

acatgtatct cagccctgtt gccatgtttc gggacccctt ccgcagagat cccaacaagc 3960

tggtgttctg tgaagttttc aagtacaacc ggaagcctgc agagaccaat ttaaggcact 4020

cgtgtaaacg gataatggac atggtgagca accagcaccc ctggtttgga atggaacagg 4080

agtatactct gatgggaaca gatgggcacc cttttggttg gccttccaat ggctttcctg 4140

ggccccaagg tccgtattac tgtggtgtgg gcgcagacaa agcctatggc agggatatcg 4200

tggaggctca ctaccgcgcc tgcttgtatg ctggggtcaa gattacagga acaaatgctg 4260

aggtcatgcc tgcccagtgg gaactccaaa taggaccctg tgaaggaatc cgcatgggag 4320

atcatctctg ggtggcccgt ttcatcttgc atcgagtatg tgaagacttt ggggtaatag 4380

caacctttga ccccaagccc attcctggga actggaatgg tgcaggctgc cataccaact 4440

ttagcaccaa ggccatgcgg gaggagaatg gtctgaagta agtagctccc tctggaccat 4500

ctttattctc atggggtgga aggcctttgt gttagggttg ggaaaagttg gacttctcac 4560

aaactacatg ccatgctctt cgtgtttgtc-ataagcctat cgttttgtac ccgttggaga 4620

agtgacagta ctctaggaat agaattacag ctgtgatatg ggaaagttgt cacgtaggtt 4680

caagcattta aaggtcttta gtaagaacta aatacacata caagcaagtg ggtgacttaa 4740

ttcttactga tgggaagagg ccagtgatgg gggtcttccc atccaaaaga taattggtat 4800

tacatgttga ggactggtct gaagcacttg agacataggt cacaaggcag acacagcctg 4860

catcaagtat ttattggttt cttatggaac tcatgcctgc tcctgccctt gaaggacagg 4920

tttctagtga caaggtcaga ccctcacctt tactgcttcc accaggcaca tcgaggaggc 4980

catcgagaaa ctaagcaagc ggcaccggta ccacattcga gcctacgatc ccaagggggg 5040

gctggacaat gcccgtggtc tgactgggtt ccacgaaacg tccaacatca acgacttttc 5100

tgctggtgtc gccaatcgca gtgccagcat ccgcattccc cggactgtcg gccaggagaa 5160

gaaaggttac tttgaagacc gccgcccctc tgccaattgt gacccctttg cagtgacaga 5220

agccatcgtc cgcacatgcc ttctcaatga gactggcgac gagcccttcc aatacaaaaa 5280

ctaattagac tttgagtgat cttgagcctt tcctagttca tgccaecccg ccccagctgt 5340

ctcattgtaa ctcaaaggat ggaatatcaa cggtcttttt attcctcgtg cccagttaat 5400

ccttgctttt attggtcaga atagaggagt caagttctta atgcctatac accaacctca 5460

tttcttttct atttagcttt ctacgtgggg gtgggagggg tagggagggg taggcgaagg 5520

gaacgtaacc acatgcttca tctcatcagg aatgccatgt ccagtaggca gagctgccac 5580

agagtgggtg tatttgtgga ggaggacttt ttcttcagga cagttaaaag agcaggtcca 5640

ctgcttggat tgacaattcc cctataggta gagagcttgc tagttcttca ggtaaaccaa 5700

ctttctattc caaatggaag ttaggtgagg agtagtggag gagttaatgc cctccatgaa 5760

gacagctcag tgtatcacct gagacagatg ggtagcccta ctgtaaaaga aggaaaagtt 5820

atttctgggt cctccattta taacacaaag cagtagtatt tttatattta aatgtaaaaa 5880

caaaagttat atatatgata tgtggatata tgtgtatttc taattcagaa accatcctag 5940

ttactgggtt tgccaagttt gaagagcttg gttaacaaga aaggatctct tgagtagagg 6000

tgggggtgca gtaccaggaa aggtggttat ctggggctca gcgctttatt actatgtggg 6060

gtttcccctg cccactctgc aggagcagat gctggacagg tagcagggtg ggacaccagt 6120

gcttgccacc acctgtccct gtgcttaggc taagatgcat atgtatccac acagagttag 6180

caggatggag ttggctggtc aacttgaaca ttgttactga taggggtggg tggggtttat 6240

tttttggtgg gactagcatg tcactaaagc aggccttttg atatattaaa ttttttaaag 6300

caaaacaagt tcagctttta atcaactttg tagggtttct aactttacag aattgcctgt 6360

ttgtttcagt gtctccatcc actttgctct tggaggaacg gaggacaggc agacctggag 6420

ttaaaacatt tgtcattttg tgtcatagtg tctactttct cccagcagaa tattcctttc 6480

cttcttagga gtcctatgga gttttgtttt-tgtttttttt ctattacgat aaacataccc 6540

cacctccatt ctggcttgcc ctgctgttct ctggttgttt gtgtgctgtc cgcagcaggc 6600

tgcctgtggt tttctcttgc catgacgact tctaattgcc atgtacagta tgttcagtta 6660

gataactcct cattgtaaac agactgtaac tgccagagca gcgcttataa atcaacctaa 6720

catttataag atttcctctt gacttgtttc tttgtggttg ggggaggaag aaaaaaaaaa 6780

gcgtgcagta tttttttgtt ccttcatttc ctatcaaaag aaaggggagt ggttctgttt 6840

tgtttactcg caaaataagc tagcttatct attggctttt cttttttttt ttttttttaa 6900

acgggctttt tcttgtacct ataatttggg gtaaggtgtg agagttttta tagttttttg 6960

agacagggtc ttggtgtata cccttggctg gcctggagct aactatgtag actgggctag 7020

cctttaactt gcagttctgc tttcaattag ggtttataca tttagtcttg gcaattccta 7080

gttccacgtt taatctcttt acatttcaaa gcagtgttat ctgaagagtt caggcgcaga 7140

gtcaattcaa tagagttaca caaaaaccta aaaaacaagt tttaaatacc aagttatgtt 7200

ggcctggcca cttttcacag ctgtccacaa ctcaatgtga caaggctaca aattggatat 7260

actagaattt cctggtgatt tggaacccct gcttcatttc ccggaaccag ggcttttggt 7320

gacagtccta gcttatcaga ttatttaaaa cagttactct tcctgccctt cttcctgaga 7380

cctttgtcca gctgccatga gccatctaca cagtacttgc ttccctgttg aagtcactga 7440

aggcacatca gcccaagaca taaaggcttg tcccggattc actagcctgg tgaacttgtg 7500

gttctctgat gttttgtcct gttttgttgt gatttagtct caaatttccc agcctggttt 7560

gaaaatctgg gctcccagcc ttcaataagg aggactacag atatgtacga ctgagccttg 7620

attccagcct catgtttata cgtctgtgct cagctccctg aaggttccag tttgaaactc 7680

aataatccag gggtcagaaa gtcttgatct tatccccaca gtatggcacc aagcctggct 7740

gagccttctg acttagtctg ccctgttgct atttaagcac ttttcttcac taggctaaaa 7800

ataaaaggag cttcctcctt tgccatggcg ctgtgcatga taggaaaagg tagctatcta 7860

ctagcatatt aactccactg tttttgcttt gtgtgtttgg tttttgagga agggtctcaa 7920

ctgtgtatcc ctggctggcc tggccggatc tagcttcgtg tcaaggacgg tgaggcgcgc 7980

caatattggc tattggccat tgcatacgtt gtatccatat cataatatgt acatttatat 8040

tggctcatgt ccaacattac cgccatgttg acattgatta ttgactagtt attaatagta 8100

atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac 8160

ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac 8220

gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt 8280

acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 8340

tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 8400

ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 8460

ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 8520

ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 8580

tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 8640

tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt 8700

tgacctccat agaagacacc gggaccgatc cagcctccgc ggccgggaac ggtgcattgg 8760

aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc tatagagtct ataggcccac 8820

ccccttggct tcttatgcat gctatactgt ttttggcttg gggtctatac acccccgctt 8880

cctcatgtta taggtgatgg tatagcttag cctataggtg tgggttattg accattattg 8940

accactcccc tattggtgac gatactttcc attactaatc cataacatgg ctctttgcca 9000

caactctctt tattggctat atgccaatac actgtccttc agagactgac acggactctg 9060

tatttttaca ggatggggtc tcatttatta tttacaaatt cacatataca acaccaccgt 9120

ccccagtgcc cgcagttttt attaaacata acgtgggatc tccacgcgaa tctcgggtac 9180

gtgttccgga catgggctct tctccggtag cggcggagct tctacatccg agccctgctc 9240

ccatgcctcc agcgactcat ggtcgctcgg cagctccttg ctcctaacag tggaggccag 9300

acttaggcac agcacgatgc ccaccaccac cagtgtgccg cacaaggccg tggcggtagg 9360

gtatgtgtct gaaaatgagc tcggggagcg ggcttgcacc gctgacgcat ttggaagact 9420

taaggcagcg gcagaagaag atgcaggcag ctgagttgtt gtgttctgat aagagtcaga 9480

ggtaactccc gttgcggtgc tgttaacggt ggagggcagt gtagtctgag cagtactcgt 9540

tgctgccgcg cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat 9600

gggtcttttc tgcagtcacc gtccttgaca cgaagcttgc cgccaccatg gtgagcaagc 9660

agatcctgaa gaacaccggc ctgcaggaga tcatgagctt caaggtgaac ctggagggcg 9720

tggtgaacaa ccacgtgttc accatggagg gctgcggcaa gggcaacatc ctgttcggca 9780

accagctggt gcagatccgc gtgaccaagg gcgcccccct gcccttcgcc ttcgacatcc 9840

tgagccccgc cttccagtac ggcaaccgca ccttcaccaa gtaccccgag gacatcagcg 9900

acttcttcat ccagagcttc cccgccggct tcgtgtacga gcgcaccctg cgctacgagg 9960

acggcggcct ggtggagatc cgcagcgaca tcaacctgat cgaggagatg ttcgtgtacc 10020

gcgtggagta caagggccgc aacttcccca acgacggccc cgtgatgaag aagaccatca 10080

ccggcctgca gcccagcttc gaggtggtgt acatgaacga cggcgtgctg gtgggccagg 10140

tgatcctggt gtaccgcctg aacagcggca agttctacag ctgccacatg cgcaccctga 10200

tgaagagcaa gggcgtggtg aaggacttcc ccgagtacca cttcatccag caccgcctgg 10260

agaagaccta cgtggaggac ggcggcttcg tggagcagca cgagaccgcc atcgcccagc 10320

tgaccagcct gggcaagccc ctgggcagcc tgcacgagtg ggtgtaata 10369

<210>4

<211>13535

<212>DNA

<213> Hamster (Hamster sp.)

<220>

<221>misc_feature

<223> seq. id.no. 1: circular plasmid GS + IgG 2A hotspot-directed vector pEE15.1 hCMVP-GFP/clone 11

<400>4

gaattcattg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 60

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 120

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 180

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 240

tgtctggcgg ccgcgacctg caggcgcaga actggtaggt atggaagatc cctcgagatc 300

cattgtgctg gcggtaggcg agcagcgcct gcctgaagct gcgggcattc ccagtcagaa 360

atgagcgcca gtcgtcgtcg gctctcggca ccgaagtgct atgattctcc gccagcatgg 420

cttcggccag tgcgtcgagc agcgcccgct tgttcctgaa gtgccagtaa agcgccggct 480

gctgaacccc caaccgttcc gccagtttgc gtgtcgtcag accgtctacg ccgacctcgt 540

tcaacaggtc tagggcggca cggatcactg tattcggctg caactttgtc atgcttgaca 600

ctttatcact gataaacata atatgtccac caacttatca gtgataaaga atccgcgcca 660

gcacaatgga tctcgaggtc gagggatctc tagaggatcc atattcgcga atatgccggc 720

atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa 780

gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc 840

ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg 900

tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag 960

agcgtcgagt cctccgtgtt cgaagcgatc cctgtccagt ggtgtgcaca ccttcccagc 1020

tgtcctgcag tctgacctct acaccctcag cagctcagtg actgtaacct cgagcacctg 1080

gcccagccag tccatcacct gcaatgtggc ccacccggca agcagcacca aggtggacaa 1140

gaaaattggt gaggaaaaca aggggagtag aggttcacaa gtgattagtc taaggcctta 1200

gcctagctag accagccagg atcagcagcc atcaccaaaa atgggaactt ggcccagaag 1260

agaaggagat actgactgtg actccctctt ggaaacttct aactatgacc acctaccttc 1320

aaggtcatga tcctctagga tagatgtcct tggtcatttc caggatcatc ctgacctaag 1380

gccataccca gggacaaagt ccctggtttg gtgccttttc tccttcaaac ttgagtaacc 1440

cccagccttc tctctgcaga gcccagaggg cccacaatca agccctgtcc tccatgcaaa 1500

tgcccaggta agtcactaga ccagagctcc acccgggaga atggtaagtg ctgtaaacat 1560

ccctgcacta gaggataagc catgtacaga tccatttcca tctctcctca tcagcaccta 1620

acctcttggg tggaccatcc gtcttcatct tccctccaaa gatcaaggat gtactcatga 1680

tctccctgag ccccatagtc acatgtgtgg tggtggatgt gagcgaggat gacccagatg 1740

tccagatcag ctggtttgtg aacaacgtgg aagtacacac agctcagaca caaacccata 1800

gagaggatta caacagtact ctccgggtgg tcagtgccct ccccatccag caccaggact 1860

ggatgagtgg caaggagttc aaatgcaagg tcaacaacaa agacctccca gcgcccatcg 1920

agagaaccat ctcaaaaccc aaaggtgaga gctgcagcct gactgcatgg gggctgggat 1980

gggcataagg ataaaggtct gtgtggacag ccttctgctt cagccatgac ctttgtgtat 2040

gtttctaccc tcacagggtc agtaagagct ccacaggtat atgtcttgcc tccaccagaa 2100

gaagagatga ctaagaaaca ggtcactctg acctgcatgg tcacagactt catgcctgaa 2160

gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa gaacactgaa 2220

ccagtcctgg actctgatgg ttcttacttc atgtacagca agctgagagt ggaaaagaag 2280

aactgggtgg aaagaaatag ctactcctgt tcagtggtcc acgagggtct gcacaatcac 2340

cacacgacta agagcttctc ccggactccg ggtaaatgag ctcagcaccc acaaaactct 2400

caggtccaaa gagacaccca cactcatctc catgcttccc ttgtataaat aaagcaccca 2460

ccaatgcctg ggaccatgta aaactgtcct ggttctttcc aaggtataga gcatagctca 2520

caggctgata tttctggcca gggttggagg acagccttgt ctataggaag agaatgaggt 2580

ttttgcactg caggactcag agctcattag ttatcctgcc ttggagtgtt ggggcttggc 2640

tttaggcagt gccttttcct tgccttccta cgaaccagca gctgccatac atagagataa 2700

tcctaggaag cctcaaatgg agaaggacac aaacccacct ccctcaggct gttcctctat 2760

cccggcccca cttctttacc taggggtttc tctgagtcta ttgtggagtt acacatggcc 2820

aggggcattc cagagaccct tgtcatccat acactcaact caggcagctt tgcacaaaca 2880

aagtctgcac acccatacag atggctcact cttgcctgtg ccatgtaggg ctgaggcaca 2940

tggctcttgc tgccccaagg gagggactat tagatagcca cactcatgct gaatcctggc 3000

ccattcaaat tagcctgctg aacaccatcc agtccatata gcacatgtat ccacatgcac 3060

gtgtgcacaa aacgcattta atacactggg-acaacaattc tgtgccctgc acagcaccta 3120

tatccagcaa tgtatcacca tacacacgac caaaaaaatt caatgcccac gtttctgcca 3180

tcacaaacag acacatcttt cctctctgtg gccactgcat tatatgctca acacaagacc 3240

tctgaagcca gatccatctc tggcacctcg gggtcatgct tcaaccccac atgaattatg 3300

caaaccatag ccataatggt ctgaatcact tcacactggg atgttcccaa gttcaggcaa 3360

gacgagccac aggctctgct gatgactgaa ggacagcaaa gggtcagtcc agctgtatag 3420

ccactgttga cctgggtcac aggccctgct gaccctccac cttctcctgt actgaaggaa 3480

tgaaagatga gacaagcata gagggcactt gaataatcca ggtcactctg aggtccaccc 3540

aaggcattat tggactcagg tgggaagctg agactggtgt cccagaggga aaggaaggaa 3600

agcaggcccc ggggagggtc tgctgtccca gtcaggctgg agatctctcc tctgaatcca 3660

tgcagacatg tctgcctcac agggaatctc tcccagcacc aaccatgttg ggacaaacac 3720

tgactgtcct ctctgttcag ggctagacct ggatgatgtc tgtgctgagg cccaggacgg 3780

ggagctggac ggcctctgga cgaccatcac catcttcatc agcctcttcc tgctcagcgt 3840

gtgctacagc gcctctgtca cactcttcaa ggttggcact gtctcccacc ctctgctgtg 3900

atggctacac tgaccacaaa atgtcctctc actcctcccc agatgtagta ggacgttact 3960

ttgctgcccc tactctgtcc cacacaccat ttcctccatt ccctgagcca tcccacattg 4020

ttctatgtga ctccacattg tgtcccatac agtctgccct tctgtctctc tggctgtcct 4080

gcgtgatcct gatactgtct tatgagacca aacctccttg cattccacac tagccttcat 4140

gaggttcaat gctgtcttac acacaatccc ctcagcctca ccatggctca aggtactctg 4200

tgagctatcc tcataccatc tccacctcaa ctcccacaat atctccactc tgacccctcc 4260

catacccagt ctcctacctg tatgaaggga attgaaggag agacaggtcg acctctgtct 4320

ttcccacaga ttggagggtc tgagcatggg cgtggtctct gactttctct cacttcccca 4380

caggtaaagt ggatcttctc ctctgtggtg gagctgaagc agacgatctc ccctgactac 4440

agaaacatga ttgggcaggg agcctaggcc acttcctctg ggatcagaag agcttcctag 4500

gccctgcaga agcccatcca tcctactgtg cagcctaaca gggaggccac actctagccc 4560

tatgactctc tgatcagaac tcccatggtc tcctctttgg aggaccacgt gcagtgcagg 4620

ctttgcccag acctaaacac ttccacagca gtcgccagat atctaactac tccggaccag 4680

aagaaccatc tccttccaaa ccagcactag ggatctgaga tctcagaatg tttgcctaag 4740

aagagctgga aatccaggct tcctgtgttc tgctacaagg acatcagcct ggatttgacc 4800

tggaccacac attttcatct aaatgagttt tccacaaagg acacgtttca gatccttgaa 4860

tgagacctct acatggaaga ccagagtcac tatacccaaa ggtcactctg tatccttgca 4920

ccagctatac tggacagctt ccttcctggt acttcagtga ccctggctga ggaaaggatc 4980

tgtgacctca actgtttgga gagcctctgg aagatgtagt cttctcttcc tgctaccacc 5040

aacatgctgg atctcagatg cagaatccaa tccacagaca ccactgacca cacaacctga 5100

agacaaggcc attgccacct ccacagagat gccatccaca ctctgtggag aaataaggag 5160

tgctttgtgc agcctctgca aagctctggc agggattaga gtatacacac tgagtactga 5220

ctaggtgacc aggcagaaaa acctccagga gaaggaacaa tgggggagag atgtgaacag 5280

atagttagaa aaagcatggt gtcacaggtc tgctctgtgg actgatttcc agattggacc 5340

acctacagca gaaaccatcg gttgcagtgg caatctagga ggaccaacct ggaataggag 5400

ggctgctgtg gtcaatggag agtagacctg tatctatttc tccactgcct cttatgacca 5460

ataagaagcc agagtctcca gacagaaaga aagaaagaaa gaaagaaaga aagaaagaaa 5520

gaaagagaga gagagagaga gagagagaga gaggaaggaa ggaaggaagg aaggaaggaa 5580

ggaaggaagg aggaggagga ggaggaggag gaggaggaga gagagagaga gagagagaga 5640

gagagagaga gagcaccagc ttttctgtga ctggaaggaa atgcttagag agcttggatc 5700

tttaaagctt cttttttcta gagaccatga atgtctttgt tctctctctc tctctctctc 5760

tctctctctc tctctctctc tctctctgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgcg 5820

tgcatgcacg ctattgtttt ggcatttgaa acaataaaac attcttttaa tattctgtat 5880

ctcatggttc cccttctgtg tggatcagcc ctaacaccca ggaacagggg acaataaaca 5940

gaccacagcc atgtacagcc ttctacctcc cttctggttc tgacctccca gaggtccctc 6000

agtgggcccc tcacagctgg gtttcttccc tggcagtgcc accaagagct caggcacctc 6060

tgagctggag gctgtcctga tgccataggc aggctatgga gcagagatga tgaccacggt 6120

ggactccagg tgagccaggc aaagcctccc atgccagaag agaagcgtgt ggtactcact 6180

ggcctcgggc tgctacggat tcagcaaaga gcatggatcg cttcgaagcc tccaagctcg 6240

acctcgggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 6300

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 6360

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 6420

gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 6480

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 6540

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 6600

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6660

tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 6720

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6780

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 6840

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 6900

aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 6960

tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 7020

cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 7080

catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 7140

ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 7200

aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 7260

ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 7320

caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 7380

attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 7440

agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 7500

actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 7560

ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 7620

ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt 7680

gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 7740

atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 7800

cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 7860

gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 7920

gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 7980

ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 8040

gacattaacc tataaaaata ggcgtatcac gaggccctga tggctctttg cggcacccat 8100

cgttcgtaat gttccgtggc accgaggaca accctcaaga gaaaatgtaa tcacactggc 8160

tcaccttcgg gtgggccttt ctgcgtttat aaggagacac tttatgttta agaaggttgg 8220

taaattcctt gcggctttgg cagccaagct agagatccgg ctgtggaatg tgtgtcagtt 8280

agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 8340

ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 8400

catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 8460

aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 8520

agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 8580

aggcctaggc ttttgcaaaa agctagcttg gggccaccgc tcagagcacc ttccaccatg 8640

gccacctcag caagttccca cttgaacaaa aacatcaagc aaatgtactt gtgcctgccc 8700

cagggtgaga aagtccaagc catgtatatc tgggttgatg gtactggaga aggactgcgc 8760

tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag aagagttacc tgagtggaat 8820

tttgatggct ctagtacctt tcagtctgag ggctccaaca gtgacatgta tctcagccct 8880

gttgccatgt ttcgggaccc cttccgcaga gatcccaaca agctggtgtt ctgtgaagtt 8940

ttcaagtaca accggaagcc tgcagagacc aatttaaggc actcgtgtaa acggataatg 9000

gacatggtga gcaaccagca cccctggttt ggaatggaac aggagtatac tctgatggga 9060

acagatgggc acccttttgg ttggccttcc aatggctttc ctgggcccca aggtccgtat 9120

tactgtggtg tgggcgcaga caaagcctat ggcagggata tcgtggaggc tcactaccgc 9180

gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat gcctgcccag 9240

tgggaactcc aaataggacc ctgtgaagga atccgcatgg gagatcatct ctgggtggcc 9300

cgtttcatct tgcatcgagt atgtgaagac tttggggtaa tagcaacctt tgaccccaag 9360

cccattcctg ggaactggaa tggtgcaggc tgccatacca actttagcac caaggccatg 9420

cgggaggaga atggtctgaa gcacatcgag gaggccatcg agaaactaag caagcggcac 9480

cggtaccaca ttcgagccta cgatcccaag gggggcctgg acaatgcccg tggtctgact 9540

gggttccacg aaacgtccaa catcaacgac ttttctgctg gtgtcgccaa tcgcagtgcc 9600

agcatccgca ttccccggac tgtcggccag gagaagaaag gttactttga agaccgcggc 9660

ccctctgcca attgtgaccc ctttgcagtg acagaagcca tcgtccgcac atgccttctc 9720

aatgagactg gcgacgagcc cttccaatac aaaaactaat tagactttga gtgatcttga 9780

gcctttccta gttcatccca ccccgcccca gagagatctt tgtgaaggaa ccttacttct 9840

gtggtgtgac ataattggac aaactaccta cagagattta aagctctaag gtaaatataa 9900

aatttttaag tgtataatgt gttaaactac tgattctaat tgtttgtgta ttttagattc 9960

caacctatgg aactgatgaa tgggagcagt ggtggaatgc ctttaatgag gaaaacctgt 10020

tttgctcaga agaaatgcca tctagtgatg atgaggctac tgctgactct caacattcta 10080

ctcctccaaa aaagaagaga aaggtagaag accccaagga ctttccttca gaattgctaa 10140

gttttttgag tcatgctgtg tttagtaata gaactcttgc ttgctttgct atttacacca 10200

caaaggaaaa agctgcactg ctatacaaga aaattatgga aaaatattct gtaaccttta 10260

taagtaggca taacagttat aatcataaca tactgttttt tcttactcca cacaggcata 10320

gagtgtctgc tattaataac tatgctcaaa aattgtgtac ctttagcttt ttaatttgta 10380

aaggggttaa taaggaatat ttgatgtata gtgccttgac tagagatcat aatcagccat 10440

accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg 10500

aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac 10560

aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 10620

tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctctag cttcgtgtca 10680

aggacggtga ctgcagtgaa taataaaatg tgtgtttgtc cgaaatacgc gttttgagat 10740

ttctgtcgcc gactaaattc atgtcgcgcg atagtggtgt ttatcgccga tagagatggc 10800

gatattggaa aaatcgatat ttgaaaatat ggcatattga aaatgtcgcc gatgtgagtt 10860

tctgtgtaac tgatatcgcc atttttccaa aagtgatttt tgggcatacg cgatatctgg 10920

cgatagcgct tatatcgttt acgggggatg gcgatagacg actttggtga cttgggcgat 10980

tctgtgtgtc gcaaatatcg cagtttcgat ataggtgaca gacgatatga ggctatatcg 11040

ccgatagagg cgacatcaag ctggcacatg gccaatgcat atcgatctat acattgaatc 11100

aatattggcc attagccata ttattcattg gttatatagc ataaatcaat attggctatt 11160

ggccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa 11220

cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt 11280

cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 11340

ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 11400

taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 11460

acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 11520

gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 11580

agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 11640

atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 11700

atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 11760

ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 11820

gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa 11880

gacaccggga ccgatccagc ctccgcggcc gggaacggtg cattggaacg cggattcccc 11940

gtgccaagag tgacgtaagt accgcctata gagtctatag gcccaccccc ttggcttctt 12000

atgcatgcta tactgttttt ggcttggggt ctatacaccc ccgcttcctc atgttatagg 12060

tgatggtata gcttagccta taggtgtggg ttattgacca ttattgacca ctcccctatt 12120

ggtgacgata ctttccatta ctaatccata acatggctct ttgccacaac tctctttatt 12180

ggctatatgc caatacactg tccttcagag actgacacgg actctgtatt tttacaggat 12240

ggggtctcat ttattattta caaattcaca tatacaacac caccgtcccc agtgcccgca 12300

gtttttatta aacataacgt gggatctcca cgcgaatctc gggtacgtgt tccggacatg 12360

ggctcttctc cggtagcggc ggagcttcta catccgagcc ctgctcccat gcctccagcg 12420

actcatggtc gctcggcagc tccttgctcc taacagtgga ggccagactt aggcacagca 12480

cgatgcccac caccaccagt gtgccgcaca aggccgtggc ggtagggtat gtgtctgaaa 12540

atgagctcgg ggagcgggct tgcaccgctg acgcatttgg aagacttaag gcagcggcag 12600

aagaagatgc aggcagctga gttgttgtgt tctgataaga gtcagaggta actcccgttg 12660

cggtgctgtt aacggtggag ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg 12720

ccaccagaca taatagctga cagactaaca gactgttcct ttccatgggt cttttctgca 12780

gtcaccgtcc ttgacacgaa gcttgccgcc accatggtga gcaagcagat cctgaagaac 12840

accggcctgc aggagatcat gagcttcaag gtgaacctgg agggcgtggt gaacaaccac 12900

gtgttcacca tggagggctg cggcaagggc aacatcctgt tcggcaacca gctggtgcag 12960

atccgcgtga ccaagggcgc ccccctgccc ttcgccttcg acatcctgag ccccgccttc 13020

cagtacggca accgcacctt caccaagtac cccgaggaca tcagcgactt cttcatccag 13080

agcttccccg ccggcttcgt gtacgagcgc accctgcgct acgaggacgg cggcctggtg 13140

gagatccgca gcgacatcaa cctgatcgag gagatgttcg tgtaccgcgt ggagtacaag 13200

ggccgcaact tccccaacga cggccccgtg atgaagaaga ccatcaccgg cctgcagccc 13260

agcttcgagg tggtgtacat gaacgacggc gtgctggtgg gccaggtgat cctggtgtac 13320

cgcctgaaca gcggcaagtt ctacagctgc cacatgcgca ccctgatgaa gagcaagggc 13380

gtggtgaagg acttccccga gtaccacttc atccagcacc gcctggagaa gacctacgtg 13440

gaggacggcg gcttcgtgga gcagcacgag accgccatcg cccagctgac cagcctgggc 13500

aagcccctgg gcagcctgca cgagtgggtg taata 13535

Claims (8)

1. A mammalian expression vector comprises a first transcription unit of a recombinant gene regulated by an mCMV promoter, and also comprises a second transcription unit containing a glutamine synthetase marker gene.

2. The vector of claim 1, wherein the mCMV promoter comprises a native transcription start point +0 to-500 bp upstream thereof.

3. The vector of claim 2, wherein the mCMV promoter comprises sequences up to the native Xho I restriction site.

4. The vector of claim 1 or 2, wherein said first transcription unit comprises an intron sequence.

5. A CHO cell transfected with the mammalian expression vector of claim 1.

6. The CHO cell of claim 5, which is a CHO-K1 cell.

7. Use of the vector of claim 1 for enhancing transfection efficiency of CHO cells.

8. The use according to claim 7, wherein the transfection is electroporation transfection or lipofection.