EFFICIENT METHOD FOR IDENTIFIABLE EXPRESSION OF NON-SELECTABLE GENES

Abstract: A recombinant plasmid vector is provided for identifiable, amplified expression of a non-selectable gene, utilizing the dominant selectable MDR phenotype.

Description: EFFICIENT METHOD FOR IDENTIFIABLE EXPRESSION OF NON-SELECTABLE GENES The present application illustrates the con¬ struction of a cloned multidrug resistance gene and various uses thereof, and provides means for linking in the same vector, a non-selectable gene desired to be expressed, with the dominant selectable multidrug resist¬ ance gene MDRl, so that the selection for MDRl reliably and simultaneously allows the amplification and over- expression of the non-selectable gene. BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accom¬ panying drawings wherein: Figure 1 shows a schematic construction of the pSKl.MDR expression vector. pHaMDRl was modified by removing pBR322 sequences between the Nrul and Sphl sites and replacing them with a Mlul linker (pHaMDR/M). pSV2CAT was modified by removing the CAT coding sequences and replacing them with a Sail linker (pSV2/Sal). The unit containing the SV40 promoter and polyadenylation signal flanking the Sal I site was removed from pSV2/Sal. Mlul linkers were added to the ends of that unit, and it was cloned into the Mlul site of pHaMDR/M. The final plasmid was designated pSKl.MDR. Grey boxes represent retroviral long terminal repeats and the MDRl open box is a human MDRl cDNA fragment coding for the multidrug transporter. The hatched box in pSV2CAT is the CAT coding region. The box designated P is the SV40 promoter + enhancer element. The box designated A con¬ tains the polyadenylation signal from SV40. Arrows indi¬ cate direction of transcription. Figure 2 is a schematic representation of pSKl.IL2R. Sail linkers were added to the ends of a DNA fragment containing the entire coding region of the p55 subunit of the interleukin-2 receptor (IL2R). The frag- ment was then cloned into the Sail site of pSKl.MDR. A clone containing the IL2R insert in the positive ("sense") orientation relative to the SV40 promoter (the direction of transcription is indicated by arrows) was designated pSKl.IL2R. A clone with the insert in the opposite orientation was called pSKl.R2LI. Figures 3A, 3B and 3C demonstrate the results of immunofluorescence. Cells transfected with pSKl.R2LI (Fig. 3A) or with pSKl.IL2R (Figs. 3B and 3C) and growing in 80 ng/ml colchicine (Figs. 3A and 3B) or in 640 ng/ml colchicine (Fig. 3C) were stained for cell-surface expression of the IL2R. Shown are representative fields from each sample, which were photographed with a Zeiss photomicroscope, 400X magnification. Figure 4 shows the results of immunoprecipita- tion. Transfected cells were labeled and immunoprecip- itations performed. The lanes marked R2LI represent lysates from control cells transfected with the pSKl.R2LI (opposite orientation) plasmid and growing at 80 or 640 ng/ml colchicine. The lanes marked IL2R are lysates from pSKl.IL2R cells, at 80 or 640 ng/ml colchicine. The numbers on the left indicate the position of molecular weight markers. The arrow points to the p55 protein specifically im unoprecipitated by anti-Tac monoclonal antibody. Figure 5 presents the results obtained from flow cytometry. Cells were prepared and analyzed using a FACScan with an argon laser (488 nm) . The x-axis repre¬ sents fluorescence intensity and the y-axis the number of cells. The thick gray line = pSKl.IL2R, 80 ng/ml colchicine; the thick black line = pSKl.IL2R, 640 ng/ml colchicine; the thin gray line = pSKl.R2LI, 80 ng/ml colchicine; the thin black line = pSKl.R2LI, 640 ng/ml colchicine. DESCRIPTION OF SPECIFIC EMBODIMENT Various objects and advantages of the present invention are achieved by the construction of a unique plasmid expression vector designated herein as pSKl.MDR which allows the expression of non-selectable genes, particularly in any suitable eukaryotic cells, some exam¬ ples of such suitable eukaryotic cells being human HeLa cells, LoVo cells, CHO cells, HT-29 cells, SK-Hep cells, A431 cells and the like. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative ony and not limiting. The terms "non-selectable" or "non-selected" gene as used herein means a gene which is desired to be expressed, but which in itself does not have a selectable phenotype. MATERIALS AND METHODS Cells NIH 3T3 cells were obtained from Dr. C. Scher, University of Pennsylvania School of Medicine and main¬ tained in Dulbecco modified Eagle medium containing 10% calf serum (Colorado Serum Co.), 5 mM glutamine, 50 U of penicillin per ml, and 50 ug of streptσmycn per ml. Colchicine was obtained from Sigma Chemical Co. and diluted from dimethyl sulfoxide stock solutions of 10 mg/ml to appropriate concentrations in complete medium. Plasmid constructions The pHaMDRl vector, described in the parent application, was first digested with Sphl and Nrul to remove sequences within the pBR322 portion of the plasmid (bp 562-972 on pBR322, including the Sail site at bp 651). Nrul leaves a blunt end and the Sphl end was made blunt with T4 DNA polymerase (Bethesda Research Labora¬ tories) . These ends were religated in the presence of Mlul linkers, creating an intermediate plasmid with the indicated pBR322 sequences replaced with a unique Mlul restriction site (pHaMDR/M) . In a separate reaction, the pSV2CAT plasmid (Gorman et al, 1982, Mol. Cell Biol. 2, 1044-1051) was digested with Hindi11 and Hpal to remove the portion of the plasmid representing the chloramphen- icol acetyltransferase gene. Hpal leaves a blunt end and the HindiII end was made blunt with the Klenow fragment of DNA polymerase I (Bethesda Research Laboratories). These ends were religated in the presence of Sail linkers, creating another intermediate plasmid (pSV2Sal) containing the desired transcription unit (the SV40 early promoter and the SV40 polyadenylation signal flanking a Sail site). pSV2Sal was digested with PvuII and BamHI to isolate the DNA fragment containing the transcription unit. PvuII leaves a blunt end and the BamHI end was made blunt with the Klenow fragment. Mlul linkers were ligated onto the ends of this transcription unit and the purified fragment was in turn ligated into pHaMDR/M which had previously been digested with Mlul. The resulting plasmid, pSKl.MDR, contains a unique Sail site for pur¬ poses of cloning foreign cDNAs within the SV40 transcrip¬ tion unit. This construction is illustrated in Fig. 1. pSKl.IL2R and pSKl.R2LI were constructed by adding Sail linkers to the ends of a 850 bp Hindlll-Xbal fragment of IL2R cDNA (Leonard et al. 1985, J. Biol. Chem. 260, 1872- 1880) which had been blunt-ended with Klenow, and then ligating the fragment into the Sail site of pSKl.MDR. pSK21.IL2R, illustrated in Fig. 2, contains the cDNA in the positive transcriptional orientation, pSKl.R2LI has the fragment in the negative orientation. A deposit of the recombinant pSKl.MDR has been made at the ATCC, Rockville, Maryland on June 14, 1989, under the accession number ATCC 68011. The deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, which¬ ever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit. Cell transfection and colchicine selection PSK1.1L2R or pSKl.R2LI DNA was transfected into NIH 3T3 cells by the calcium phosphate coprecipitation method and cells were selected in the presence of colchicine as described by Kane et al (1988) Mol. Cell. Biol. 8, 3316-3321. Resulting colonies were pooled into 80 ng/ml colchicine and the concentration of colchicine was increased stepwise up to 1 g/ml to select for ampli¬ fied expression of the transferred MDR cDNA. Immunoprecipitations Cells were labeled with 100 uCi of [ 35 S]methionine (Amershal Corp.) per ml in Dulbecco-Vogt medium lacking methionine (National Institutes of Health Media Unit) and supplemented with 5% calf serum, 5 mM glutamine, 50U of penicillin per ml, and 50 g of strep¬ tomycin per ml. Labeling was for 15 hrs and cells were lysed in RIPA buffer [20 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Aprotinin] . Immunoprecipita¬ tions were performed essentially as described by Kane et al, supra, Gal et al (1985) J. Cell Biol. 100, 535-544, using 5 x 10 6 trichloroacetic acid-precipitable counts of cell lysate and anti-Tac, a mouse monoclonal antibody specific for the 55 kDa IL-2 receptor (Uchiyama et al, 1981, J. Immunol. 126, 1393-1397; Leonard et al, 1983, Proc. Natl. Acad, Sci. USA 80, 6957-6961). Secondary incubations were performed for 2 hrs at room temperature (about 22-24°C) with Protein A-Sepharose [20% in phosphate buffered saline (PBS) lacking calcium and magnesium], followed by washes as described by Gal et al, supra. Immunoprecipitates were run on a SDS-10% polyacrylamide gel as described by Laemmli (Laemmli, U.K., 1979, Nature (London) 227, 680-685). The gel was treated with 2,5-diphenyloxazole in dimethyl sulfoxide, and fluorography was performed at -70°C (Bonner et al, 1974, Eur. J. Biochem. 46, 83-88). Immunofluorescence Populations of cells growing in 80 ng/ml colchicine or 640 ng/ml colchicine were seeded into 35 mm tissue culture dishes. Cells were stained live, first with the anti-Tac monoclonal antibody, followed by goat anti-mouse antibody conjugated to rhodamine (Jackson Immuno Research). Antibody incubations were for 30 in. at 4°C. The anti-Tac antibody concentration was 10 g/ml and the anti-mouse antibody was 50 g/ml. Both were in PBS with calcium and magnesium plus 2 mg/ml bovine serum albumin (BSA) . After the final antibody incubation, cells were washed and then fixed for 10 min. at room temperature with 3.7% formaldehyde. Flow Cytometrv Flow cytometric analysis of the IL2R cell- surface marker was performed as follows. Cells were stained indirectly by incubating with the anti-Tac anti¬ body followed by goat anti-mouse antibody conjugated with fluorescein (Jackson ImmunoResearch) . Incubations were for 30 min. at room temperature, in PBS without calcium or magnesium plus 1% BSA. After the secondary antibody incubation, cells were fixed for 10 min. in 3.7% formaldehyde, washed, and resuspended at a final concentration of approximately 1 x 10° cells per ml of PBS. Flow cytometry was performed using a FACScan (Becton-Dickinson) with an argon laser (488 nm) . Surface receptor quantitation was done using a method described by LeBouteiller et al (1983) J. Immunol. Methods 61, 301- 305, Monograph: Fluorescent Microbead Standards (1988) Flow Cytometry Standards Corporation, with standards purchased from Flow Cytometry Standards Corporation. Data analysis was done with FACScan Research software (Becton-Dickinson) . Percent positive cells was estimated by determining the fraction of cells falling into a win¬ dow of fluorescence intensity chosen to exclude 99% of the negative control cells. RESULTS Construction of pSKl.MDR Expression Vector The pHaMDRl expression vector was modified by inserting a transcription unit consisting of the SV40 early region promoter and enhancer and the SV40 poly¬ adenylation signal, flanking a unique Sail restriction site. The scheme for constructing this new plasmid, pSKl.MDR, is illustrated in Figure 1, and the details of the construction have been described herein above. An intermediate form of pHaMDRl was prepared by removing sequences within the pBR322 portion of the plasmid (pb 562-972 on the pBR322 map, including the Sail site at position 651) and replacing them with a unique Mlul site (pHaMDR/M) . A DNA fragment containing an SV40 transcrip¬ tion unit, engineered with a Sail site between the SV40 promoter and the polyadenylation sequences, was ligated into the Mlul site of pHaMDR/M. The resulting plasmid, pSKl.MDR, has the SV40 transcription unit inserted into the pBR322 region of the parent plasmid. The Sail site is unique and can be used as a cloning site for inserting foreign cDNAs. The direction of transcription for the MDRl gene and for the SV40 transcription unit are indi¬ cated by arrows in Figures 1 and 2. Expression of a Foreign cDNA in pSKl.MDR To test pSKl.MDR in transfection experiments, a cDNA for the interleukin-2 receptor (IL2R) was cloned into the Sail site of the SV40 transcription unit. The plasmid containing the IL2R (pSKl.IL2R) was transfected into NIH 3T3 cells by the calcium phosphate coprecipita- tion method (Shen et al, 1986, J. Biol Chem. 261, 7762- 7770). As a control, the same cDNA was inserted into pSKl.MDR in the opposite transcriptional orientation (pSKl.R2LI) and transfected in parallel. Cells were selected for the ability to grow in 60 ng/ml of colchicine. The initial analysis was performed on trans¬ fected cells which had been selected in 60 ng/ml of colchicine for two weeks and then pooled into the same selective medium for an additional 9 days. These popula¬ tions were analyzed by immunofluorescence for the presence of IL2R surface antigen. An estimated 75-80% of the cells which were transfected with the pSKl.IL2R con¬ struction expressed significant amounts of IL2R on the cell surf ce (data not shown) . The control cells trans¬ fected with pSKl.R2LI and selected in 60 ng/ml colchicine were completely negative for the IL2R antigen. Amplification of MDRl and IL2R Mixed populations of resistant cells (either pSKl.IL2R or pSKl.R2LI) were pooled into medium with 80 ng/ml of colchicine and then grown in increasingly higher concentrations of colchicine, up to 640 ng/ml, to amplify the expression of the transfected genes (Kane et al, supra) . During this amplification process, the original population of cells growing in 80 ng/ml colchicine were maintained in culture at that same level of drug. By the time the final sets of analyses were performed, popula¬ tions at 80 ng/ml colchicine had been in culture for 3- 3.5 months, while the cells at 640 ng/ml had been growing at that drug concentration for 1-1.5 months. These groups of cells were then analyzed by three different methods to determine the level of IL2R expression in each group: immunofluorescence, immunoprecipitation and FACS analysis. The results of the immunofluorescent staining are shown in Figure 3. Most of the cells in both the 80 ng/ml and the 640 ng/ml populations of pSKl.IL2R trans- fectants expressed IL2R on the cell surface. However, there were more cells in the low drug than in the high drug that appeared to express little or no cell surface IL2R. In addition, there was a higher percentage of cells expressing large amounts of IL2R in the 640 ng/ml culture than in the 80 ng/ml culture. There were, how¬ ever, a significant number of cells in the 80 ng/ml popu¬ lation which stained very brightly for the cell surface IL2R antigen. Thus, even after 3 months in culture, under relatively mild selective conditions, some cells expressed a very large amount of non-selected gene (IL2R). Again, the control pSKl.R2LI cells were com¬ pletely negative for IL2R staining. To quantify better the expression of IL2R by each of these cell populations, immunoprecipitations were performed. Populations of cells were labeled meta- bolically with 35 S-methionine and IL2R was precipitated from whole cell extracts using the anti-Tac monoclonal antibody. As shown in Figure 4, cells growing in 640 ng/ml colchicine synthesized significantly more IL2R protein, on average, than cells in 80 ng/ml colchicine. Flow cytometry was used to determine the dis¬ tribution of IL2R expression among cells in the high-drug and low-drug populations (Figure 5). Cells in each pop¬ ulation were reacted with the anti-Tac antibody and then with FITC-conjugated goat anti-mouse antibody. The curves in Figure 5 show number of cells (y-axis) versus fluorescent intensity (x-axis) and thus represent the distribution of the level of cell-surface IL2R expression within the respective populations. The results of this analysis indicate that even after long-term culturing at 80 ng/ml of colchicine, greater than 80% of the drug-resistant cells also expressed IL2R. Although there was a broad range of the level of IL2R expression, a significant number of these cells expressed very high amounts of the protein on their cell surface. When compared with standards of known IL2R number, the average number of receptors per cell in this population was about 280,000. At 640 ng/ml colchicine, on the other hand, the level of IL2R expression was more uniformly at the high end of the range. This population had an average of 740,000 receptors/cell. Therefore, after selecting cells in increasing concentrations of colchicine, there was an enrichment for cells which were very drug resistant and which profusely expressed IL2R. The net result was a coordinate increase in expression of the MDRl cDNA and of the non-selected gene (cell-surface receptor) . In short, the results presented herein using the IL-2 receptor as a test gene, clearly demonstrate significant expression of the protein (IL2R) under the control of the SV40 promoter. Expression was efficiently amplified by growing the transfected cells in high con¬ centrations of selective agent. The MDRl gene has the advantage of being easily selectable in a variety of cell types. In addition, the amplification process proceeds readily and rapidly. Since the MDRl gene product is a cell surface molecule, it provides a means for selecting transfected cells via fluorescence-activated or magnetic bead cell sorting. Furthermore, the MDRl system can be used either by cotransfecting the pHaMDRl plasmid and a non-selectable gene present on a separate plasmid, or by including the desired cDNA within the transcription unit of pSKl.MDR as demonstrated here, or by fusing the non-selected protein to the carboxy terminus of P-glycoprotein (Germann et al, 1989, J. Biol. Chem.). Of course, having demonstrated herein the simultaneous overexpression of a non-selectable IL2R cDNA with the selectable MDRl cDNA, other non-selectable genes can be similarly coexpressed in suitable transfectable cells as will be suggested to one of ordinary skill in the art. A number of selective agents other than colchicine, such as vinblastine, adriamycin and the like can also be conveniently employed. It is understood that the examples and embodi¬ ments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A recombinant plasmid expression vector containing MDRl cDNA and a site for the insertion of a non-selectable gene, wherein when said non-selectable gene is inserted in said site in the expression vector, and a cell is transfected with the plasmid vector comprising said non-selectable gene, said non-selectable gene is amplified and overexpressed simultaneously with the MDRl cDNA. 2. A plasmid vector of claim 1 having the characteristics of ATCC deposit number 68011. 3. A plasmid vector of claim 1 or 2, further comprising a non-selectable gene inserted at said site. 4. A plasmid of claim 3, wherein the non- selectable gene is an interleukin 2 receptor gene. 5. A cell transfected with a plasmid vector of claim 1. 6. A method for identifiable expression of a non-selectable gene, comprising inserting the cDNA of a non-selectable gene desired to be expressed into the plasmid expression vector of claim 1, transfecting said expression vector into cells and identifying the expression of said non-selectable gene in said cells by the dominant selectable MDR phenotype with which said non-selectable gene is simultaneously overexpressed. 7. A method for ampli ied production of a gene product, comprising transfecting suitable cells with a plasmid expression vector of claim 1 and recovering the product overexpressed by a non-selectable gene by conventional isolation and purification means.

Inventors: GOTTESMAN MICHAEL MARC, KANE SUSAN ELIZABETH, PASTAN IRA HARRY, GOTTESMAN, MICHAEL, MARC, KANE, SUSAN, ELIZABETH, PASTAN, IRA, HARRY
Applicants: US HEALTH, THE UNITED STATES OF AMERICA, REPRESENTED BY THE S
Application year: 1990
Main classification: C07H15
IPC codes: C07K14, C12N5, C12N15, C12P21, C12R1