CA2076975A1

CA2076975A1 - Mammalian expression vectors

Info

Publication number: CA2076975A1
Application number: CA 2076975
Authority: CA
Inventors: Kevin W. Moore; Paul J. Zavodny
Original assignee: Individual
Current assignee: Merck Sharp and Dohme LLC
Priority date: 1990-02-28
Filing date: 1991-02-26
Publication date: 1991-08-29
Also published as: AU7444191A; WO1991013160A1; EP0517768A1; JPH05503015A

Abstract

This invention provides novel recombinant plasmids which can be used to clone heterologous genes in E. coli and to clone and express such genes in mammalian cells. These plasmids contain a number of unique restriction sites that can be used to facilitate the deletion or replacement of genetic elements and the insertion of heterologous genes in the plasmids. The nature and placement of the unique sites is such that regeneration of the sites following endonuclease cleavage can readily be achieved with a minimum of manipulative effort. Host cells containing the recombinant plasmids are also provided by this invention.

Description

WO 91/13160 PCI`/US91/01078 ```` 2~76~7~

MAMMALIAN EXPRESSlOt~l VECTORS

BACKGROUND OF THE !NVEI~ION

Much of the worl< in the recombinant DNA field to the present has focused on the use of bacterial host cells such as E ~!i ~or the production of recombinant polypeptides and proteins. Yet, the use of bacterial cells has a number of undesirable aspects. For example, most proteins and polypeptides produced in E coli accumulate in the cytosol or periplasmic space. Recovery of these gene products requires disruption o~ the cells and, often, extraction with a detergenl or chaotropic agent such as urea or guanidine-hydrochloride. Proteins thus recovered are frequently in incorrectly folded forms and are difficult ,; ~ to purify because they must be isolated from the numerous other cellular constituents. Furthermore, bacteria cannot carry out glycosylation which is needed to complete the synthesis of many ~ interesting gene products and are frequently incapable of forming the specific disulfide bonds which are essential for the proper conformation and biological activity of many eukaryotic proteins.

To overcome these deficiencies of bacterial expression systems, the attention of genetic engi~eers is increasingly turning to the use of eukaryotic host cells. Cells such as yeast and mammalian cells can secrete desired gene products into the culture medium and can carry out essential glycosylation processes as well. Yet, the use of ;

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mammalian cells for recom~inant DNA cloning and expression also presents a host of technical obstacles that must be overcome. For example, the endogenous plasmids that have proven to be so useful in bacteria are usually not replicated by higher euka~otic cells. As a result, other approaches must be taken.

One approach has been to use the lower eukaryotic yeast, ~accharomvces cerevisiae, which can be grown and manipulated with the same ease as E. coli. Yeast cloning systems are available, and through the use of such systems the efficient expression in yeast of a human interferon gene has been achieved [Hitzeman et al., Nature (London) ~:717 (1981)]. It has been found, however, that yeast cells do not correctly transcribe at least one heterologous mammalian gene that contains introns, the rabbit ~-globin gene [Beggs et al., Nature - 15 (London) ~:835 (1980)3.

In another approach, heterologous genes have been inserted into mammalian cells by means of ~irect uptake. This has been accomplished, ~or example, by calcium phosphate co-precipitation of - 20 cloned genes, by which procedure about 1-2% of the cells can generally be induced to take up the DNA. Such a low level of uptake, however, produces only a very low level of expression of the desired gene product. Where mammalian cells can be ~ound which lack the thymidine kinase gene (tk- cells), better results can be obtained by co- -transformation. ~k- cells, which cannot grow in selective HAT
(hypoxanthine-aminoterin-thymidine) medium, can regain this lost enzymatic activity by taking up exogeneous DNA (such as herpes simplex viral DNA) containing the tk gene through calcium phosphate co-precipita~ion. Other DNA covalently ligated to the tk DNA or merely mixed with it will also be taken up by the cells and will o~ten be co-;~ expressed lsee Scangos et al., Gene 14:1 (1981)].

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A number of inves~igators have developed expression vectors for use in eukaryotic cells. For example, Okayama et al. [Mol.
Cell. Biol. 3:~80 (1983)] have developed a ~pcD~ vector system which incorporates Simian virus 40 (SV4G) control elernents. Kaufman ~U.S.
Paten~ No. 4,740,461 ) has described eukaryotic cell vectors which incorporate SV40 elements and a selectable marker, for co-amplification. These vectors are said to be useful, e.g., for the production of tPA in CHO cells. Choo et al. [DNA ~:529 (19~6)] -ave developed two recombinant vectors for the direct expression an-amplification of cDNA in cultured mammalian cells. Pfarr et al. [DNA
4:461 (1985)] have developed a ~modular" vector, pDSP1, which contains two independent mammalian transcription cassettes.

~ A variety of genetic engineering techniques are employed ;; 15 in the construction of such recombinant vectors. General methods have 7~ been described by Cohen et al. (U.S. Patent No. 4,237,224), Collins et .i al. (U.S. Patent No. 4,304,863) and Maniatis et al. (Molecular Cloning: A
Laboratory Manual, 1982, Coid Spring Harbor Laboratory).
. Panayotatos [Gene ~1:291 (1984)] has disclosed lhe complete end-filling of restriction site overhangs to generate new sites upon ligation, following the cleavage of palindromic restriction sites. Korch [Nuc. Acids Res. 15;3199 (1987)] and Hung ~ Nuc. Acids Res. 12:1863 (1984)]
have described ways to manipulate restriction endonuclease sites involving partial end-fllling, to permit the joining of DNA fragments which ,~. 2~ normally cannot be ligated together.
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SUMM~13Y O~E~Y~

This invention provides recombinant plasmids which can be used to clone heterologous genes in F. ~Q!i and to clone and express such genes in a variety of mammalian cells. The plasmids are ~ characterized by strategically placed genetic elements and unique '~!, restriction sites, the nature of which facilitates the excision and/or ..~
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2~7~97~ -4-substitution of elements and the restoration of the original restnction sites.

The recombinant plasmids of the invention comprise DNA
5 sequences which, in the direction of transcription, contain:

(a) a ~-lactamase gene and a pBR322 origin of replication delimited by an Xbal and a B~lll restriction site, (b) an SV-40 or SRoc promoter delimited by a ~dlll and an Xhol restriction site, (c) a polylinker containing one or more unique restriction sites delimited by a E~l and an E~RI restriction site, and (d) a polyadenylation signal sequence delimited by 1~ an EÇQRI and an 2~k~1 restriction site. ~ .
Preferably, the polyadenylation signal sequence is a human ,B-globin or an SV-40 early or late region polyadenylation signal sequence.

Some of the recombinant plasmids of the invention further comprise a dihydrofolate reductase (DHFR) transcription unit comprising, in the direction of transcription, a mouse mammary tumor -~ virus long terminal repeat (MMTV-LTR) promoter, a DHFR cDNA, a splicin~ signal sequence and a polyadenylation signal sequence.
2~ Preferably, the splicing signal sequence is a small t antigen intron and the polyadenylation signal sequence is an SV-40 early region polyadenylation signal sequence.

When present in the plasmids, the DHFR transcription unit ,; 30 is positioned between the DNA sequences containing the ,B-lactamase gene and pBR322 origin of replication and the SV-40 or SR promoter.
The DHFR transcription unit is delimited, in the direction of transcription, by a ~111 and a ~ dlll restriction site. The DNA sequence containing , :' .

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the MMTV-LTR promoter within the transcription unit is delimited by two ~9lll restriction sites.

In other embodiments of the invention, the recombinant plasmids comprise DNA sequences containing a ~-galactosidase (~-gal) or a chloramphenicol acetyltransferase (CAT) transcription unit in place of the DHFR transcription unit. When present, these sequences are also terminated by a 13~111 and a ~dlll restriction site.

In still other embodiments, a splicing signal sequence is present between the DNA sequences containing the SV-40 or SRa promoter and the polylinker and is delimited, in the direction of transcription, by an Xhol and a e~II restriction site. This splicing signal sequence is preferably a 16S/19S intron.

BRIEF ~RIPTION OF THE FIGURES

This invention can be more readily understood by reference to the accompanying Figures in which: -~- 20 , Fig. 1 is a schematic representation ot recombinant plasmid pDSVS, showing unique restriction sites and elements.
., Fig. 2, panels A-H, is a schematic representation showing the construction of plasmid pDSVS from various starting plasmids.
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Fig. 3 is a schematic representation of plasmid pDSRS.
This plasmid is similar to pDSVS except for the substitution of an SRcL
promoter for the SV-40 promoter of pDSVS.
'i 30 Fig. 4 is a schematic representation of plasmid pSRS. This i~ plasmid is similar to pDSRS except that the DHFR transcr3ption unit has been deleted in pSRS.

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,,, WO 91/13160 PCI/US91/010'18 2~7~75 Fig. ~, panels A and B, is a schematic representation of the construction of plasmids pDSRG and pSRG. These plasmids are similar to pDSRS and pSRS, respectively, except that plasmids pDSRG and pSRG contain a human ~-globin polyadenylation signal sequence 5 instead of an SV-40 late region polyadenylation signal sequence.

Fig. 6, panels A-C, is a schematic representation of the construction of plasmid pGSRG-hlL5, which is capable of directing the production of recombinant human interleukin-5 in mammalian cells.

L)E~cRlpTloN OFTHE INVENTION

All references cited herein are hereby incorporated in their entirety by reference.
~i As used herein, the term ~transcription~ means the synthesis of messenger RNA (rnRNA) from a DNA template. A
"promoter" is a DNA sequence that directs the binding of RNA
polymerase and thus ~promotes~ transcription. "In the direction of 20 transcription~ means from upstream to downstream (i.e., from 5' to 3'~, or in the direction of movement of RNA polymerase as it carries out the transcription of DNA. The SRa promoter used in this invention has been described by Takebe et al. [Mol. Cell. Biol. 8:466 ~1988)]. The SV-40 promoter has been described by Okayama et al. [Mol. Cell. Biol. ~:280 25 (1983)]-An "enhancer" is a DNA sequence that can potentiate the transcription of a gene without regard to the nature of the gene.
Although promoters must be positioned upstrearn of genes that are to be 30 expressed, the positioning of enhancers is not critical. They can function whether upstream or downstream of a gene, and even when they are inverted with respect to the other DNA sequences (i.e., in the 3' to 5' orientation).

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"Splicing signais~ are DNA sequences which can also enhance the transcription of genes encoding some proteins. An ~origin of replication~ is a point in a plasmid at which replication of the plasmid by a host cell is initiated.
A "polyadenylation signal sequence~ is a DNA sequence located downstream of the translated regions of a gene at which adenine ribonucleotides are added to form a polyadenylate tail at the 3' end of the mRNA.

A "polylinker" is a DNA sequence which contains one or more unique restriction sites. Polylinkers facilitate the insertion of heterologous genes (i.e., genes not normally present in a host cell) that are to be expressed in the plasmids of the invention. As used herein, 15 the term heterologous "gene~ includes both isolated genes and cDNAs prepared from mRNA.

. "Restriction sites" are DNA sequences which definecleavage points for restriction endonucleases. Such sites are "unique"
when only one of a given site is present in a plasmid. The plasmids of ,,! this invention may contain unique 2~ QRI, Smal, ~mHI. ~LI, ~I, -~ Xhol and Hindlll sites.
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In the most basic embodiments, the plasmids of the invention comprise, in the direction of translation:
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(a) a ,B-lactamase gene and a pBR322 origin of replication delimited by an Xbal and a ~glll restriction site, ' (b) an SV-40 or SRa promoter delimited by a ~:_dlll and an Xhnl restriction site, (c) a polylinker containing one or more unique restriction sites delimited by a ~II and an ~QRI restriction site, and ,.
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With a heterologous gene inserted into the polylinker, elements (b) - (d) together comprise a heterologous gene transcription unit.

The ,B-lactamase gene encodes an enzyme which degrades the antibiotic ampiciliin. Because of this activity, the presence of the ,B-lactamase gene in a host cell confers on the cell resistance to ampicillin in the culture medium. As a result, host cells harboring the plasmids of the invention can be selected by growth in such medium, while other cells lacking such resistance will not survive. In the accompanying figures, the presence of the ~-lactamase gene is indicated by"AMP" or"AMPr.

A recombinant plasmid containing only the foregoing elements can be used for the transient expression of a heterologcus gene inserted into the polylinker. Insertion of a gene into the polylinker .~. 20 is facilitated by the presence in the polylinker of unique restriction sites, including ~I, ~LI, ~HI, ~m~l and çQRI sites. Gen~ insertion is ~ easily achieved if the termini of the gene following isolation arecompatible with the ~II and/or ~11, ~mHI or ~QRI sites. If they are not, they can readily be modi~ied using standard methods to be compatible. Alternatively, the gene termini can simply be blunt ended by standard methods and inserted into the ~ cleaved plasmid, because ~l digestion produces blunt-ended cleavage.
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The polylinker need not contain all of the restriction sites , 30 mentioned above; any undesired sites can be deleted by standard methods. Conversely, other sites can be added if need be, as long as they are unique sites so that cleavage with the corresponding restriction ^ endonuclease does not also cut the plasmid elsewhere.
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g In other embodiments of the invention which can be used for transient expression, a ,B-gal or CAT transcription unit is inserted between the DNA sequences containing the ~-lactamase gene and pBR322 origin of replication and the S~/-40 or SRa promoter. Both the ,B-gal gene and the CAT gene are well known in the art, the former having been described by Hall et al. [~. Mol. Appl. Gen. ~:101 (1983)], the latter by Gorman et al. [Mol. Cell. Biol. 2:1044 (1982)]. When present in the plasmids, these genes provide useful marker activities for transformanVtransfectant screening.
All of the foregoing plasmids are useful for transient expression in mammalian cells, but longer-term expression cannot be achieved with such plasmids. That is because there is no selective pressure to maintain the plasmids in host cells. Over time, they are lost.
1 S For more prolonged expression, the plasmids of the invention preferably contain a DHFR transcription unit in place of a ~-gal or CAT transcription ,?,~ unit. The elements of the DHFR transcription unit have been described by Lee et al. [Nature ~:228 (1981)].

The presence of a DHFR transcription unit in the plasmids provides two advantages. Firstly, when introduced into a host cell lacking dihydrofolate reductase (~, into CHO-dhfr cells), the unit ? provides a means of selection. Cells harboring the plasmids can easily be selected from other cells in medium lacking hypoxanthine and , 25 thymidine. Secondly, cells containing high levels of the dhfr cDNA will .
survive culture in medium containing high levels of methotrexate, an inhibitor of ~ novo purine synthesis, while cells expressing low levels of the cDNA will not. Such conditions will thus enable selection for cells ~''? which have increased or amplified the number of copies of the dhfr gene.
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;l When a heterologous gene that is to be expressed is also present in a plasmid with the dhfr gene, expression of the heterologous gene will be co-amplified as the host cell makes many copies of the dhfr ~., ~ .
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2~7697~ 10-gene. In the examples below, culturing cells harboring a plasmid containing the DHFR transcription unit in the presence of methotrexate caused increased production of both dihydrofolate reductase and interleukin-5 (IL-5).

In the examples below, an SV-40 late region [Okayama et al., Mol. Cell. Biol. 3:280 (1983)] or human ~-globin [Lawn et al., Cell 21:647 (1980)] polyadenylation signal sequence was used downstream of the polylinker. These sequences are interchangeable and can also 10 be used in the DHFR transcription unit in place of the SV-40 ~arly region sequence (Lee et al., supra). The SV-40 early region sequence could also be used downstream of the polylinker. The only requirement for such substitutions is that the sequences used in such positions in the plasmids must be terminated by the restriction sites indicated in Fig. 1, if 15 it is desired to later remove the polyadenylation signal sequence. Since the nucleotide sequences of all three are known, they can be chemically . synthesized andtor adapted for such use using conventional methods.

The plasmids of the invention may optionally contain 20 enhancer regions. Useful enhancers are obtained from animal viruses such as SV-40, polyoma virus, bovine papilloma virus, cytomegalovirus, retroviruses or adenovinuses. Ideally, the enhancer used should be from a virus for which the host cells are permissive (i.e., from a virus which normally infects cells of the host type).
The enhancer regions of a number of viruses are known ,. [see, ~,g" Luciew et al., Cell 33:70~ (1983)]. It would be a routine matter to excise these regions based upon published restriction maps for a virus selected and, if necessary, to modify the termini to enable splicing 30 the enhancer into the recombinant plasmids. Alternatively, the enhancers can be synthesized from published sequence data.

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~7~975 The only requirement for a selected enhancer is that it preferably not contain a restriction site(s) that would destroy the uniqueness of the above-mentioned sites. One of skill in the art could readily determine this from the published sequence data and the known 5 restriction site sequences. Although the enhancers from many viruses can be used, the enhancer from SV-40 (Takebe et al., ~upra) is - preferred for use in this invention. Although the location of the : enhancers within the plasmids is not critical, the ~Qdlll restriction site is a convenient point for insertion.

. A wide range of heterologous genes can be inserted into the plasmids, including but not limited to genes encoding blood clotting or fibrinolytic enzymes, regulatory proteins such as the various Iymphokines or hormones, growth factors, oncogene products and soluble or cell-surface antigens and receptors. One of the plasmids of the invention has been used to express a gene encoding a soluble form of a gamma interferon receptor. In the examples below, the use of another plasmid to express a gene encoding IL-5 is illustrated.
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.~, 20 Such genes will often contain wild-type translated ~ sequences encoding prepro-polypeptides (~, secretory leaders), `i which may be desirable for the structure, stability arid/or secretion of the mature proteins. Such polypeptides are removed during post-.. translational processing by the host cells. Alternatively, such prepro-25 sequences can be deleted by well known methods prior to insertion of . the genes into the plasmids. The genes may also contain 5' and/or 3' noncoding sequences.

. The genes or cDNAs to be expressed must contain 30 translaSion initiation and stop codons, although the SV-40 or SRc~
promoter used provide the necessary transcription initiation signals.

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~7~7~ - 12-Although the preferred order of elements in the plasmids is as described above, any element inserted upstream of the promoter for the heterologous gene, such as the ~-gal or CAT transcription unit or the DHFR transcription unit, can be inserted downstream of the heterologous gene transcription unit instead. This can conveniently be done by the use of the Xbal restriction site.

The placement of the genetic elements and unique restriction sites within the plasmids facilitates the substitution of elements or the removal of elements for use in otner plasmids or vectors.
It also permits easy restoration of the original restriction sites after cleavage has been carried out.

For example, the DHFR transcription unit is easily 1~ removed. ~II/KIenow followed by ~dlll/exoVII regenerates the ~!ll site. ~liDdlll/Klenow followed by ~lll/exoVII regenerates the ~dlll site.
Treatment of the polyadenylation signal sequence in the E~RI/Xbal fragment by 2~1/E~QRI double digestion followed by Klenow i polymerase and religation excises the sequence and regenerates both sites. 2~h~1/Klenow followed by ~llexoVII and religation removes the Xhol/E~I fragment splicing signals and regenerates an 2~hQI site.

~he plasmids of the invention can be used in many mammalian host cells, including primary explants from various tissues.
The use of established and/or transformed cell lines, however, is ' preferred. Cell lines that can be used include but are not limited to the African green monkey kidney (COS), Chinese hamster ovary (CHO), NS-1, SP210, NIH 3T3, NIH 3T6, C127, CV-1, HeLa, mouse L and Bowes cell lines. Where co-amplification using the DHFR transcription -- 30 unit is desired, dihydrofolate reductase-deficient cells such as CHO-dhfr . cells lUrlaub et al., Proc. Natl. Acad. Sci. USA ~:4216 (1980)] are preferred.

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Plasmid DNA can be introduced into mammalian cells in a number of ways. Transient expression studies in mammalian cells such as COS cells can be carried out using the DEAE~extran method with chloroquine boost [Yokota et al.,Proc. Natl. Acad. Sci. USA ~:68 (1985)3. Transfection of suspension cells (~g., NS-1) can be accomplished using the method of electroporation ~Potter et al., Proc.
Natl. Acad. Sci. USA 81:7161 (1984)]. Fibroblasts such as CH0 cells can be transfected using the ca.cium phosphate precipitation method [Graham et al., Virology 52 4~6 (1973)]. All three of these methods are described below in detail.

Identification of cells harboring the plasmids can be made using standard methods. For ~xample, host cell genetic material can be probed by Southern blot analysis. Expression of the desired heterologous gene can be detected by standard immunochemical or enzymatic arialysis, or by bioassay.
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EXAMPLE~

!n the examples that follow, percentages for solids in solid mixtures, liquids in liquids, and solids in liquids are given on a wVwt, volhol and wVvol basis, respectively, unless otherwise indicated. Sterile conditions were maintained during cell culture.
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General Methods Small scale isolation of plasmid DNA ~rom saturated ovemight cultures was carried out according to the procedure of Birnboim et al. [Nuc. Acids Res. 7:1513 (1979)]. This procedure allows the isolation of a small quanti~y of DNA from a bacterial culture for analytical purposes. Unless otherwise indicated, larger quantities of ~, plasmid DNA were prepared as described by Clewell et al. [J. Bacteriol.
110:1135 (1972)3.

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Specific restriction enzyme fragments derived by the cleavage of plasmid DNA were isolated by preparative electrophoresis in agarose followed by electroelution (Maniatis et al., ~" p. 164).
Gels measuring 9 x 5 1/2 cm were run at 50 mA for 1 hour in Tris-Acetate buffer (Maniatis et al., ~, p. 454) and then stained with 1 llg/ml ethidium bromide to visualize the DNA. Appropriate gel sections were excised and melted at 65C for 10 minutes and then diluted with 5 ml of a low salt buffer containing 0.2 M NaCI, 20 mM Tris-HCI (pH 7.4) and 1 mM EDTA. The DNA was then concentrated using a Elutip-D column (Schleicher and Schuell Inc., Keene, NH) following the manufacturer's instructions and precipitated at -20C with ethanol in the presence of 10 1l9 of yeast tRNA carrier (Bethesda Research Laboratories, Bethesda, MD).

The restriction enzymes, DNA polymerase I (Klenow fragment) and T4 DNA ligase were products of New England ~iolabs, Beverly, MA, and the methods and conditions for the use of these enzymes were essentially those of the manufacturer. T4 DNA ligation was carried out for 16 hours at 4C in a buffer containing 50 mM Tris-HCI, pH 7.8, 10 mM MgCI2, 20 mM dithiothreitol, 1 mM ATP and 50 ' mg/ml bovine serum albumin. Klenow blunt-ending of single-stranded DNA ends was carried out in restriction enzyme buffer which had been adjusted to contain 1 mM dGTP, dATP, dCTP and TTP.

~hçmica! Synthesis Oligonucleotides used as linkers were either purchased from New England Biolabs, Beverly, MA, or prepared by standard methods using a Model 380A DNA synthesizer (Applied BioSystems, 30 Foster City, CA).

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TransforrnatiQn and TransfQctiQn E ~!i cultures were transformed essentially as described by Maniatis et al., ~.

For transient expression in COS cells, the plasmid DNAs were introducted by DEAE-dextran treatment with a chloroquine boost rlokota et al., Proc. Natl. Acad. Sci. USA ~:68 (1985)]. Briefly, 10 ml of a suspension of COS cells were plated onto 100 mm culture dishes at 10 approximately 105 cells/ml in DME medium, penicillinlstreptomycin (pen/strep) and 2 mM glutamine (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). After the cells reached approximately 50% confluence, the medium was removed and the cells were washed with 10 ml of senJm-free DME.

A plasmid DNA-DME-DEAE-dextran mixture was prepared as follows: for each plate, DME medium (4.2 ml) supplemented with ` 50 mM Tris-HCI, pH 7.4, glutamine and antibiotics as described above was mixed with 80 ~l of DEAE-dextran (20 mg/ml in sterile, distilled 20 water; Pharmacia, Piscataway, NJ). Plasmid DNA (10 ~,lg/dish) was - added to 4.2 ml of the DME-DEAE~extran mixture, and the mix~ure was l added to the washed cells. Each 100 mm culture dish was incubated for 4 hours, the DNA solution was aspirated from the plate, and the plate was washed with 5 ml of seNm-free DME. After the addition of 5 ml of 25 chloroquine solution (150 IlM chloroquine in DME, with 7% fetal bovine serum), the cells were incubated for 3 hours. After this incubation, the chloroquine was removed by aspiration and the plate washed with 5 ml `~ of DME and aspirated. Each plate then received 10 ml of DME (10%
fetal bovine serum, antibiotics and glutamine as above) and was 30 incubated for approximately 72 hours. The cells were collected or the conditioned medium was harvested for bioassay.
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For stable transfection of myeloma cells, the plasmid DNAs were introducted by electroporation [Potter et al., Proc. Natl. Acad. Sci.
USA 81 :7161 (1984)]. Briefly, approximately 10 ml of myeloma cells growing in suspension in complete DME medium supplemented as above (5 x 10~ cells/ml) were centrifuged and resuspended in 1 ml of complete DME. Plasmid DNA (40 ~19) was added to the 1 ml suspensinn and the cell/DNA mixture was divided into 0.25 ml aliquots. Each aliquot of cells was placed in an electroporation chamber and shocked with an electric pulse of 300 volts and 960 microFd capacitance using a Gene 10 Pulser (Bio-Rad, Rockville Centre, NY). The cells were held at room temperature for 10 minutes and then the four aliquots were combined and placed in a plastic 175 tissue culture flask in a total volume of 10 ml of complete DME. After a 3-day incubation period, the cells were diluted with selection medium to a final concentration of 0.5 ,ug/ml mycophenolic 15 acid (BRL/Life Technologies, Gaithersburg, MD), 100 llg/ml Xanthine (Sigma, St. Louis, MO), 15 llg/ml Hypoxanthine (Sigma3, 10% fetal bovine serum and 880 ~,Ig/ml glutamine. The cells were diluted to give approximately 1,000 cells/0.2 ml/well of a 96-well tissue culture plate.
Colonies of transfected cells could usually be seen forming in 7-14 days.
20 Cells were collected or conditioned medium was harvested for assay as described above.
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;' The calcium phosphate precipitation method was used for the stable transfection of fibroblasts with plasmid DNAs. The protocol 25 included in the Mammalian Transfection Kit (Stratagene, LaJolla, CA) was followed. Briefly, fibroblasts were plated into a 100 mm tissue culture dish at ~ x 105 cellsldish in complete DME medium prepared as described above plus 1X nonessential amino acids (100X stock from Gibco), and 4 ~g/ml thymidine and 15 ~lg/ml hypoxanthine (both 30 contained in 1 ûOX stock frsm Gibco). A~er a 24 hour incubation, 10 ~19 of plasmid DNA (as a calcium phosphate precipitate) were added per `~ plate. The cells were incubated for 24 hours, and the plates were ` washed with serum-free DME medium and aspirated.

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The cells were incubated for 24 hours, split 1 10 and allowed to grow for 24 hours before the addition of selection medium.
The selection medium contained DME, glutamine and nonessential amino acids as described above, plus 5% dialyzed fetal bovine serum (Gibco) without antibiotics. After colonies began to form, medium containing methotrexate was added stepwise until cells were obtained which could grow at a 1 ,uM level of methotrexate. This produced amplification of the integrated locus [Kaufman et al., J. Mol. Biol. ~:601 (1982)]. Cells were harvested or conditioned medium was collected for 10 assay as described above.

Cell Cultures ~- ~Q!i strain C600 (ATCC 23724) was used for plasmid 15 constn~ctions. The cells were grown in 20:10:5 TYE medium (20 9 tryptone, 10 g yeast extract, ~ 9 NaCI per liter).

~; COS cells (ATCC CRL 16~0) were cultured in DME
medium (Gibco, Grand Island, NY) plus antibiotics, 2 mM glutamine (~iibco) and 10% fetal bovine serum (Hyclone, Logan, UT) as described above. NS-1 cells (ATCC TIB 18) were cultured in DME medium like the COS cells. GHO dhfr cells (clone DXB-11 ) were obtained from Dr. L.
Chasin, Columbia University, NY. These cells were routinely carried in ;i complete DME medium with added 1X nonessential amino acids, 25 thymidine and hypoxanthine (from 100X stock, Gibco).

ConstnJction of Plasmid pDSVS

'!i Some of the starting plasmids and elements contained in 30 them haYe been described by Lee et al. [Nature ~ 228 (1981)], ` Zavodny et al. lJ. Interferon Res. 8:483 (1988)] and Okayama et al. lMol.
Cell. ~iol. ~:280 (1983)~.

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The unique E~!ll site of plasmid pMTV-dhfr was destroyed by digestion with ~lll followed by fill-in of the protruding 5' overhangs with DNA polymerase Klenow fragment to form plasmid pM~V-DHFR. A
DNA fragment containing the murine dihydrofolate reductase cDNA and SV-40 early region poiyadenylation signal was obtained by elution of the large ~QRl/~l~l DNA restriction fragment of pL23 followed by restriction with ~indlll.

The resulting ~indlll/EcoRl fragment of pL23 (containing the SV-40 polyadenylation signal and dhfr cDNA) was inserted into EcoRl/~ndIII-digested plasmid pMT11 S to form plasmid pL12R-A. The unique ~dllI site of plasmid pL1 2R-A was converted to a ~g!ll site by Klenow polymerase fill-in of the Hindlll-generated 5' overhangs followed by ligation of Bqlll linkers (CAGATCTG; No. 1036, New England Biolabs), .~QIll digestion, ligation and transformation of F. ~QIi strain C600 to form plasmid pL13R-B. The unique E~amHl site of plasmid .:
pL13R-B was converted to a ~ndlll site by Klenow polymerase fill-in of the ~mHl-generated 5' overhangs followed by ligation of ~indlll linkers ~ (CMGCTTG; No. 1022, New England Biolabs), ~lindlll digestion, ,~ 20 ligation and transformation of . coii strain C600 to form pL1 4R.
The fragment containing the pcD-derived SV-40 splicing ? signal sequence and murine gamma interferon cDNA was removed from plasmid pMgamma18 by 2~ digestion, and the plasmid backbone was . 25 religated to form plasmid pL27B. The DNA fragment containing the ' SV-40 early promoter and SV-40 late region polyadenylation signai of .~ plasmid pL27B was removed by ~l restriction and Klenow polymerase ' fill-in of the 5' overhangs, ligation of f~l linkers (GCTGCAGC; No. 1024, New England Biolabs), and E~l/~Qdlll simultaneous digestion. The resulting ~ dlll/E~l fragment containing the SV-40 DNAs described above was ligated to ~indllll~l-di9ested pL14R to generate plasmid pL15R-B. The E~mHl restriction site immediately adjacent to the unique ~indlll site of pL15R-B was removed by ~mHl partial digestion to ~, generate linearized DNA. This was followed by Klenow polymerase fill-.i , , :
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in of the 5' overhangs and religation of the plasmid to generate plasmid pL602.

The small EcoRI/~I fragment of pL602 was removed by E~oRI/e~II digestion and replaced with a synthetic DNA containing compatible EcoRI/E~I overhangs but which resulted in the loss of both sites, due to lack of reconstitution of the complete recognition sequence.
In addition, the synthetic DNA contained the recognition sequence for Xbal. The nucleotide sequences of the synthetic DNAs, AB176 and AB177, are shown in Fig. 2C. The resulting plasmid, pL603, lacked the RI/~I fragment of pL602 and contained a unique 2~.al site in its place.
The SV-40 late region polyadenylation signal was removed from plasmid pCDV-1 by ~l/2~kQI digestion, Klenow 15 polymerase fill-in of the 5' overhangs, ligation to EcoRI linkers (GGAATTCC; No. 1020, New England Biolabs), EcoRI digestion and insertion into E~RI-digested plasmid pMT11 S to form plasmid pKMP-1.
The unique Xhol site of plasmid pKMP-1 was removed by Xhol digestion, Klenow polymerase fill-in of the 5' overhangs and religation to 20 form plasmid pKMP-2.
. . , The DNA fragment containing the SV-40 early promoter and intron from plasmid pL1 was removed by ~11 digestion, S1 Nuclease digestion of the 3' overhang, Klenow polymerase fill-in (of any 5' overhangs produced by S1 digestion), ligation of 2~1 linkers (CTCTAGAG; No. 1032, New England Biolabs), and simultaneous r~striction with ~l and ~ dlll. The resulting ~lindlll-2~LI-linkered fragment was inserted into ~Indlll/2~1-restricted plasmid pMT11 S to form plasmid pKML-1. The DNA fragment containing the SV-40 early prornoter and intron was removed from plasmid pKML-1 by ~lindlll/B9111 digestion and inserted into ~ndlll/~nHI-digested pUC13 to create .,~!, plasmid pUCL1-E. The overhangs created by ~mHI and ~ll are identical, sothe DNA~ragmentsannealed. Both sites, however, were . ~
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WO 91/13160 PCI`/US91/01078 2~7~7~ -20-destroyed because the entire recognition sequences ~or both enzymes were lost.

The ~mHI site of pUCLl -E was destroyed by ~mHI
5 digestion, Klenow polymerase fill-in of the 5' overhangs and religation to create pUCL2-A. The SV-40-containing ~indlll/Xbal fragment ol pU(::L2-A was inserted into ~!lndlll/2~k~1-cut plasmid pKMP-2 to generate plasmid pcDL-4. The ~!ll and ~I sites of plasmid pcDL-4 were destroyed by Bglll/2~1 restriction followed by exonuclease Vll digestion 1 0 of the protruding 5' overhangs and religation to generate plasmid pL64G. The Xhol/~mHI fragment of plasmid pL64G containing the SV-40 intron was inserted into 2~bQI/BamHl-digested pL603 to generate plasmid pT443-2.

1 5 The unique Ndel site in plasmid pcDV-1 was converted to an Xbal site by ~1~1 digestion, Klenow polymerase fill-in of the 5' overhangs, ligation to 2~11inkers (CTCTAGAG; No. 1032, New England Biolabs), Xbal digestion and religation to form plasmid pT512-2. The SV-40 polyadenylation signal-containing DNA fragment of 20 plasmid pT443-2 was replaced with the SV-40 polyadenylation signal-containing fragment of pT512-2 by ~mHI/Xb~l digestion and ligation to form plasmid pT~14-2. A synthetic DNA fragment containing restriction sites for .BamHI, ~im~l and E~QRI (the original BamHI site of pT51 4-2 was lost on cloning) was inserted between the BamHI and ~!l sites of : 25 plasmid pT~14-2 to create plasmid pT519-1. The nucleotide sequences of the DNAs used, AB199 and AB200, are shown in Fig. 2G.
The two ~lindlll sites of plasmid pL23 were converted to ~ll sites by ~indlll digestion, Klenow fill-in of the 5' overhangs on both DNA
fragments, ligation of E~lll linkers (CAGATCTG; No. 1036, New England `~ 30 Biolabs), digestion with J~lll and ligation of the DNA mixture to create plasmid pL17-A.

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Finally, the DNA fragment containing the MMTV-LTR
promoter was removed from plasmid pLT7-A by digestion with ~gLII and inserted into the unique ~lll site of plasmid pT519-1, to oreate the general eukaryotic expression vector pDSVS.

Construction of Plasmid ,QPSRS

To replace the SV-40 promoter in pDSVS with an SR(x promoter [Takebe et al., Mol. Cell. Biol. 8-466 (1988)], the SRa promoter was excised from plasmid pcDL-SRa296 by digestion with ~lndlll and and E~ 1. The DNA fragment containing the SRa promoter was electroeluted from an agarose gel and ligated to ~Ldlll/~l - digested pDSVS to generate pDSRS. This construction is shown schematically inFig.3.

~onstruction of ~lasrQ~ pSRS
/
Synthetic oligonucleotides AB361 and AB362 (Fig. 4) were synthesized as described above. When annealed, these oligonucleotides produced a double-stranded DNA with a B~lll - overhang at one end and a ~Lndlll overhang at the other. The synthetic ; DNAs were annealed by heating to 95C in a volume of 20 microliters and then allowed to reach room temperature.

The general expression plasmid pDSVS was digested with ~111 and ~lindlll and electroeluted from an agarose gel. The .~ annealed oligonucleotides were ligated to E~~ iQd~ di9ested pDSVS
to produce the plasmid pSVS. The result was the removal of the dhfr ' transcription unil, with the regeneration of both restriction sites. The SRa promoter was then substituted for the SV40 promoter of pSVS.
The SRa promoter was isolated from plasmid pcDL-SRa296 by digestion with ~lindlll and 2~h~1. The promoter element was ligated to d~ hQl-di9ested pSVS to generate plasmid pSRS.
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Construction of Plasmids pDSflG and p~RG

Syn~hetic oligonucleotides AB523 and AB524 (Fig. 5) were - -synthesized as described above. When annealed, these olibonucleotides introduced restriction sites for Xhol, BamHI, ~lll, and ~!1 between the existing EcoRI and ~indlll sites of pUC19. The AB523 and AB5~4 synthetic oligonucleotides were annealed as above and ligated to E~QRI/~j~ldlll-digested pUC19 to produce plasmid p679-2.

The human beta globin 3' untranslated region was ~` obtained by ~mHI digestion of plasmid pBut-7 and ligated to ~digested p679, to produce plasmid p658J. This plasmid was used at the source for the human beta globin polyadenylation signal in the SRa promoter containing expression plasmids. Plasmid p658J was digested with ~indlll, end-filled with DNA Klenow fragment, the Klenow was inactivated, and the plasmid was digested with ~QRI. This fragment was ligated to 2~i-digested, Klenow-treated and ~QRI-digested pDSRS and pSRS, to produce pDSRG and pSRG, respectively.
.~ .
~ 20 ConstnJction of Plasmid pGSRG-hlL5 :
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Human IL-5 cDNA was removed from plasmid pcD-SRo~-hlL5 by the polymerase chain reaction (PCP~) [Saiki et al., - -:! Science ~:487 (1988)]. The forward primer, AB790 (Fig. 6), was designed to introduce ~ll restriction site upstream of a modified initiation region representing an optimum Kozak configuration [Kozak, J.
~,~ Cell. Biol. 108:229 (1989)]. The reverse primer, AB791 (Fig. 6), was designed to introduce an EcQRI restriction site downstream of the termination codon for human IL-5. The directions of the prirners are ~, 30 shown by the arrows in Fig. 5. After 30 cycles of PCR (95C for 2 min., 40C for 2 min. and 72C~ for 2 min.) the amplified DNA ~ragment was digested with Sall/Jiindlll and ligated to ~~ indlll-digested ,oSRS to produce the expression plasmid pSRS-hlL5. The expression cassene ,. for human IL-5 containing the SRa promoter, splice sites, and hlL-5 .3 . ~ :`
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't, WO 9t/13160 PCI`/US91/01078 "` 2~6~75 cDNA up to the E~RI site was removed from plasmid pSRS-hlL~ by ~indlll/~QRl digestion and ligated to ~jndlll/EcoRl~igested pDSRG
and pSRG, to produce pDSRG-hlL5 and pSRG-hlL5, respectively. The dominant marker, - Qli xanthine-guanine phosphoribosyltransferase 5 (gpt), was then introduced into the hlL-5 expression plasmid pSRG-hlL5.
The region containing the E. coli. gpt transcription unit, a portion of the rearranged immunoglobulin in the heavy chain variable region (Vh1), amplicillin marker and F. coli plasmid origin of replication was removed from plasmid pD8Ch2UK by digestion with Xhol and Sall, end-fill with 10 Klenow polymerase as above, inactivation of the Klenow enzyme and digestion with 2~k~1- The region from 2~1 to ~all containing the elements described above is identical to the 2~1 to Sall region in the plasmid pSVD8tbeta [Schnee et al., Proc. Natl. Acad. Sci. USA ~.:6904 (1987)]. This 2~,1/~l fragment was inserted into ~jndlll-digested, 15 K!enow treated and ~l~igested pSRG-hlL5 to produce pGSRG-hlL5.
This human IL-5 expression plasmid will express human IL-5 using the SRa promoter, SV40 splice signals and human beta globin 3' untranslated region.

Interleukin-5 Bioassay The erythroleukemic human cell line TF-1 was obtained from Dr. T. Kitamura (Tokyo University), although the murine BCL1 cell line (ATCC TIB 197) described by Dutton et al. lJ. Immunol. 13?:2451 (1984~] could have been used instead. TF-1 cells were routinely cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum, 1 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF), and pen/strep. For bioassay, the cells were washed twice in phospate buffered saline (PBS, Gibco) and aliquoted into 96-well plates ~` 30 in RPMI 1640 at 4 x 104 cells/well. An equal volume containing test samp!e in RPMI 1640 and 10% fetal bovine sen~m was added to the cells. The cells were incubated for 48 hours at 37C, 10 ~Lg of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, . ~ .
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2 ~ 7 ~ 24 -Sigma) dye dissolved in PBS were added to each well, and the cells were incubated for 6 hours.

A 1:1 dilution in each well was made using acid-5 isopropanol ~0.1M HCI:95% isopropanol), and the contents of the wells were thoroughly mixed. The colorimetric assay o~ Mosmann [J.
Immunol. Methods ~5:55 (1983)] was carried out using a V-max spectrophotometer (Molecular Devices, Palo Alto, CA) set at 570 nm.
Blank absorbance at 630 nm was subtracted from the measurements at 10 570 nm. Activity (expressed in units/ml) was defined as the inverse of the dilution of the sample required to give half-maximal proliferative induction of TF-1 cells when cultured for 48 hours. ~ -Lls~ of eDSYS Derivatives tQ Produce Recombinant Human IL-5 The Human IL-5 expression plasmids described above were transfected into three different cell lines and the conditioned media from the cultures were assayed for human IL-5 biological ac~ivity, using the TF-1 cell bioassay. Conditioned medium from COS cells was 20 prepared as described above. Recombinant CHO cells, either unamplified or selected to grow in 1 ,uM methotrexate, were seeded into 5 ml of DME medium (plus 10% fetal bovine serum, 1X nonessential amino acids and 880 ~Lg/ml glutamine without antibiotics) a 5 x 105 cells/ml and cultured for 72 hours. Cell culture conditioned medium was 25 harvested and passed through a 0.22 ',1 Nalgene low protein binding syringe filter in preparation for bioassay. Recombinant myeloma cells (NS-1, ATCC TIB 18) were seeded into 5 ml of HB101 medium (Hana s; Biological, Alameda, CA) supplemented with 800 llg/ml glutamine at - 5 x 105 cells/ml and cultured for 72 hours as described above for CHO
30 cells. After incubation, the conditioned medium was harvested for bioassay, with the results shown in Table 1.

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- 2~-Tabl,e I
Expression Level (units/ml~ Qf Hurnan IL-5 Expression Pla~mid COS cells CHO Cell~a CHO cellsb pSP~S-hlL5 2,000 n.d. n.d. n.d.
pDSRG-hlL5 n.d. <2,000 20,000 n.d.
pSRG-hlL5 2,000 n.d. n.d. n.d.
pGSRG-hlL5 n.d. n.d. n.d. 12,000 .

a Plasmids were unamplified.
b Plasmids were amplified in 1 tlM methotrexate.
n.d. = not de~ermined.

The data of Table I show that human IL-5 was efficiently produced in all three cell types. The IL-5 biological activity in the ,~ conditioned media ranged from 2,000 units/ml in COS cells ~transient , 20 expression) to 20,000 units/ml in amplified CHO cells. Production in the CHO cells increased more than ~en fold upon selection with methotrexate. The unamplified production of IL-5 in the NS-1 cells, in ' view of the variability of the bioassay used, was rou~hly comparable to the amplified production in the CHO cells.
'.~ 25 Plasmid Deeosits ;, Plasmids pDSVS, pDSRS, pDSRG, pSRS and pSRG were deposited February 21, 1990 with the American Type Cu!ture Collection, , ' 30 Rockville, MD, under the provisions of the Budapest Treaty on the ' Interna~ional Recognition of the Deposit of Microorganisms for the ,, ~, ., :
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~, , Purposes of Patent Procedures. They have been assigned Accession Nos. ATCC 68231, 68232, 68233, 68234 and 68235, respectively.

Many modifications and variations of this invention may be 5 made without departing from its spirit and scope, as will become apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A recombinant plasmid comprising DNA sequences which, in the direction of transcription, contain:

(a) a .beta.-lactamase gene and a pBR322 origin of replication delimited by an XbaI and a BgIII restriction site, (b) an SV-40 or SR.alpha. promoter delimited by a HindIII
and an XhoI restriction site, (c) a polylinker containing one or more unique restriction sites delimited by a PstI and an EcoRI restriction site, and (d) a polyadenylation signal sequence delimited by an EcoRI and an XbaI restriction site.

2. The recombinant plasmid of claim 1 which further comprises a DHFR transcription unit comprising, in the direction of transcription, an MMTV-LTR promoter, a DHFR cDNA, a splicing signal sequence and a polyadenylation signal sequence, which transcription unit is between the .beta.-lactamase gene and pBR322 origin of replication and the SV-40 or SR.alpha. promoter and is delimited by a BgIII and a HindIII
restriction site.

3. The recombinant plasmid of claim 1 which further comprises a .beta.-gaI transcription unit between the .beta.-lactamase gene and pBR322 origin of replication and the SV-40 or SR.alpha. promoter, which .beta.-gaI transcription unit is delimited by a BgIII and a HindIII restriction site.

4. The recombinant plasmid of claim 1 which further comprises a CAT transcription unit between the .beta.-lactamase gene and pBR322 origin of replication and the SV-40 or SR.alpha. promoter, which CAT
transcription unit is delimited by a BgIII and a HindIII restriction site.

5. The recombinant plasmid of any one of claims 1 to 4 which further comprises a splicing signal sequence between the SV-40 or SR.alpha. promoter and the polylinker, which splicing signal sequence is delimited by an XhoI and a PstI restriction site.

6. The recombinant plasmid of any one of claims 1 to 5 which further comprises a viral enhancer.

7. The recombinant plasmid of claim 1 which is pDSVS.

8. The recombinant plasmid of claim 1 which is pDSRS.

9. The recombinant plasmid of claim 1 which is pSRS.

10. The recombinant plasmid of claim 1 which is pDSRG.

11. The recombinant plasmid of claim 1 which is pSRG.

12. The recombinant plasmid of claim 1 which is pGSRG-hIL5.

13. The recombinant plasmid of claim 1 which is pDSRG-hIL5.

14. An E. coli bacterium harboring the recombinant plasmid of any one of claims 1 to 13.

15. A mammalian cell harboring the recombinant plasmid of any one of claims 1 to 14.