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for the expression of foreign genes. Recent years have witnessed an explosion of interest in this area with the development of better methods ofgene transfer and the identification of increasingly powerful promoters. Indeed, one may legitimately ask whether the pendulum has not swung too far in this direction, to the point where people may inappropriately dismiss the potential of alternative host systems (see [2] in this volume). Seen as a whole, the decision as to which host to utilize should be dictated not primarily by technical considerations--the engineering of all three host systems discussed herein has become too routine for t h a t - - b u t by considerations of a scientific nature. We have accordingly sought to communicate a sense of the advantages and disadvantages of using mammalian cells as hosts with respect to this standard. The protocols presented in Section V of this volume should be sufficiently detailed to execute a successful expression strategy. [39] Vectors Used for Expression

in M a m m a l i a n

Cells

By RANDAL J. KAUFMAN Introduction of Genetic Material into Cells The techniques of gene isolation, modification, and transfer into appropriate host cells have provided a means to understand protein structure and function by enabling the production of large amounts of proteins that previously could be isolated in only minute quantities, and by allowing the generation of proteins with specific, designed alterations. The advantages of using mammalian cells as a host for the expression of a gene obtained from a higher eukaryote stem from experience that the signals for synthesis, processing, and secretion of these proteins are usually properly and efficiently recognized in mammalian cells. There are many purposes for the use of mammalian cells for expression of foreign genes: (1) To confirm that genes isolated by a variety of different approaches can direct the synthesis of a desired protein; (2) to evaluate the effect of specific mutations introduced into a gene; (3) to isolate genes directly based on screening or selecting recipient cells for the production of a particular protein; (4) to produce large amounts of proteins that are normally available in only limited quantity; and (5) to analyze the physiological consequences of expression of specific proteins in mammalian cells in order to study biological regulatory controls. Two major criteria in evaluating what type of expression vector to use for mammalian cells are (1) the method desired for introducing the foreign gene into the mammalian cell, and (2) the control elements to be utilized for efficient mRNA expression and protein syntheMETHODS IN ENZYMOLOGY, VOL. 185

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form ~erved.

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sis. This article first discusses the advantages, utilities, and disadvantages of various host-vector systems available for transfer and expression or heterologous genes in mammalian cells. Then the genetic control elements for efficient expression are discussed. Finally, approaches to monitor efficient vector function are described. H o s t - V e c t o r Systems for Heterologous G e n e Expression Two general types of methods for transfer of genetic material into mammalian cells are those that are mediated by virus infection and those that are mediated by direct DNA transfer. Many viruses which infect mammalian cells have evolved mechanisms to usurp the protein synthesis machinery of the host to produce their viral proteins. The ability to engineer the genetic material of these viruses has made it possible to insert desired coding regions under the control of the viral expression elements and to produce infectious virus particles to obtain high levels of foreign gcnc expression. Viral-mediated gene transfer provides a convenient efficient means to introduce foreign DNA into the majority of the recipient cells. In addition, for many viruses, viral replication yields multiple copies of template DNA which can serve to amplify transcription of the foreign gene. Since some viruses can infect a wide range of cell types derived from different species, viral-mediated gene transfer may allow the convenient introduction of foreign genes into a variety of different cells. This article addresses those viral vector systems which have demonstrated success, which are used more frequently, or which have significant potential. For a more detailed review of the different eukaryotic viral vectors, see Ref. I.

Viral VectorSystems Papovaviruses. Papovaviruses are small, nonenveloped DNA-containing viruses of which SV40 and polyoma are the best studied, z SV40 virus replicates in simian cells and polyoma virus replicates in murine cells. The SV40 viral genome of approximately 5 kilobases (kb) is divided into an early region which encodes the transformation antigens, large T and small t antigens, and the late region which encodes the viral capsid proteins VP1, VP2, and VP3. After viral infection the early genes are expressed. After 12 hr, viral DNA replication is initiated and, at 36-48 hr, optimal expression of the late genes occurs. Newly replicated viral genomes are then assembled into progeny virus. About 72 hr postinfection, the cells detach and die. N. Muzyczka (ed.), Curr. Top. Microbiol. Immunol. in press (1990). 2 j. Tooze (ed.), "DNA Tumor Viruses." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981.

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Since SV40 replicates to a higher copy number than polyoma virus, SV40 has been more widely utilized for successful heterologous gene expression. Foreign DNA can be inserted into either the early or late regions of SV40 to generate defective viral genomes. Replicating virus stocks can be prepared by providing a helper virus to complement the deficient region. Alternatively, it is possible to prepare helper-free recombinant viruses by propagation of the virus on appropriate cell lines which express the essential missing viral function. For example, African green monkey kidney CV- 1 cells transformed with an origin-defective SV40 mutant virus (thus COS cells), express the SV40 large T antigen. 3 Large T antigen is required in trans to replicate DNA containing the SV40 origin of replication. COS cells can efficiently support the episomal replication of bacterial plasmids containing the SV40 origin of replication. This ability to replicate and amplify transfected DNA to greater than 100,000 copies/cell has provided a very efficient means of obtaining high-level transient expression of DNA directly introduced into these cells. Eventually the transfected cells die, presumably due to the very high level of DNA replication. The transient expression observed after plasmid DNA transfection of COS cells is probably the most efficient and convenient means to obtain expression of foreign genes (see below). Since COS cells express T antigen, they can also complement recombinant SV40 viruses which contain early region replacements. Most success with SV40 recombinant viruses has been obtained by gene replacements into the late region of SV40. High expression levels (greater than 10s molecules/cell) have been obtained by insertion of eDNA into the 5' untranslated region of the SV40 late mRNAs and deleting the genes for VPI, VP2, and VP3. 4 Two vectors for this purpose are pSVLHA 4 in which genes can be introduced between the HpalI and BamHI sites, and pspLT5, 5 in which genes can be introduced between the XhoI and BamHI sites. In order to complement such SV40 late gene replacements, cells are transfected with an equal amount of DNA from an SV40 mutant in the early region such as d11055 ~ or DNA from another SV40 recombinant containing an early gene replacement such as pSV40-rlnspBR322. 6 For complementation, SV40 early region replacements are recommended since they minimize the potential for recombination to generate wild-type viruses.

3 y. Gluzman, Cell23, 175 (1981). 4 M.-J. Gething and J. Sambrook, Nature (London) 292, 620 (1981). 5 X. Zhu, G. M. Veldman, A. Cowie, A. Carr, B. Schaflhausen, and R. Kamen, J. ViroL 51, 170 (1984). M. Horowitz, C. L. Cepko, and P. A. Sharp, in "Eukaryotic Viral Vectors" (Y. Gluzman, ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, p. 47. 1982.

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The preparation of virus stocks has been described. 4,7 The SV40 recombinant DNA is prepared as bacterial plasmid DNA. The plasmid sequences are removed by restriction digestion and the DNA is then ligated at low concentrations (1 pg/ml) to generate closed circular DNA molecules. The ligated DNA is mixed with the complementing virus DNA prepared in a similar manner and transfected into monkey CV-I cells (obtained from ATCC) using the DEAE-dextran transfection protocol as described below for COS-1 cells. Since the percent of cells that acquire DNA is usually less than 10%, it is necessary to prepare a primary virus stock and passage this once in order to obtain a high-titer virus stock that can be used subsequently to infect all the recipient cells. To prepare a high-titer stock, transfected cells are harvested by scraping into the growth medium at 5 days posttransfection. The cells in the suspension are lysed by quick freeze-thawing 3 times (from dry ice-ethanol to 37 ° water bath). One-half milliliter of the stock may then be used to infect a new 100-mm dish of CV-1 cells. Five days postinfection, the cells are again harvested and lysed as described above in order to obtain the high-titer virus stock. Twentyfour hours prior to infection, CV- l cells are subcultured 1 : 4. For infection, the growth medium is removed and 0.3 to 0.5 ml of the virus stock is applied to the cells for l hr at 37 °. The cells are then rinsed and fed with fresh medium. Generally, the cells or conditioned medium are harvested at 48 hr postinfection. Although high expression levels (1 - 10/~g/106 cells) make SV40 recombinant viruses attractive, multiple disadvantages have limited their use. First, the range of permissible cell types is limited to monkey cells. Second, viral infection results in cell lysis after 3 to 4 days, necessitating only transient studies and limiting large-scale processes to a batch harvest procedure. Third, the requirements for viral packaging restrict the size of the foreign DNA which can be inserted. Generally, SV40 can accommodate up to 2.5 kb of foreign DNA inserted into either the early or the late region. Fourth, DNA rearrangements frequently occur during replication of these viruses. Finally, not all genes introduced into SV40 viruses behave the same. Since the reason for the variability is not understood, it is not possible to predict the probability of success. SV40 recombinant viruses may prove more useful for the expression of potentially toxic proteins. Vaccinia Virus. Vaccinia virus is a member of the poxvirus family) Vaccinia replicates in the cytoplasm of mammalian or avian cells. A linear double-stranded DNA genome of 185 kb is packaged into the virus core. Vaccinia encodes its own transcription and RNA processing system within 7 C. Doyle, J. F. Sambrook, and M.-J. Gething, J. Cell Biol. 100, 704 (1985). s B. Moss, in "Virology" (B. N. Fields and D. M. Knipe, eds.), p. 2079, 2nd Ed. Raven, New York, 1990.

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the viral particle. On infection, about 100 genes are expressed early and host protein synthesis is shut off. After DNA replication at 6 hr postinfection, about 100 late genes are expressed. There is no evidence of splicing for any vaccinia virus transcript. Virus is formed about 6 hr after infection and continues for about 2 days. Because of the large size of the vaccinia genome, DNA must be inserted by homologous recombination. Plasmid vectors have been constructed in which foreign DNA can be inserted under control of either a vaccinia early or late promoter and flanked by DNA from a nonessential region of the vaccinia genome. After transfection of cells that have been infected with vaccinia virus, the foreign DNA recombines into the viral genome. Most commonly, insertion is directed into the thymidine kinase gene such that the TK- phenotype can be used for selection of recombinants. 9 Alternatively, inclusion of the fl-galactosidase gene within the expression plasmid permits a selection for recombinant plaques by screening with an appropriate color indicator. 1° Recombinant viruses may also be screened by DNA hybridization or antibody binding. A vaccinia virus/bacteriophage T7 promoter vector system utilizes the efficient and selective T7 bacteriophage RNA polymerase to express mRNA in a mammalian cell. A recombinant vaccinia virus (vTF7-3) is used to express the T7 RNA polymerase.t i Foreign DNA is cloned between two fragments of T7 DNA containing the ~bl0 promoter and the T~b termination sequences into a plasmid vector (pTF7-5) containing cloning sites and flanking vaccinia virus thymidine kinase gene sequences for homologous recombination. Transfection of the plasmid DNA containing the desired coding region insert into cells infected with the T7 polymerase vaccinia recombinant virus yields high-level expression of the foreign gene from the T7 promoter. As an alternative to transfection, the T7 promoter and desired gene can be introduced into another vaccinia virus and higher expression obtained by coinfection of the two recombinant viruses, one encoding the RNA polymerase and the other encoding the desired gene under control of the T7 promoter. With this coinfection approach, it is possible to introduce the desired gene into 100% of the recipient cells. Coinfection can yield very high levels of mRNA derived from the T7 promoter (about 30% of the total cell RNA) at 24-48 hr postinfection. ~2 However, only 5-10% of the RNA is properly capped and the RNA is 9 D. Panicali and E. Paoletti, Proc. Natl. Acad. ~'ci. U.S.A. 79, 4927 (1982). io S. K. Chakrabarti, K. Breehling, and B. Moss, Mol. Cell. Biol. 5, 3403 (1985). n R. R. Fuerst, E. G. Niles, F. W. Studier, and B. Moss, Proc. Natl. Acad. Sci. U.S.A. 83, 8122 (1986). 12 T. R. Fuerst, P. L. Earl, and B. Moss, Mol. Cell. Biol. 7, 2538 (1987).

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poorly translated. Current research is directed to identify means to increase the translation of the T7-derived transcript. The expression level obtained with this system when successful is approximately 3% of the total protein by using either a strong late promoter of vaccinia or the vaccinia/T7 hybrid system. The major use for the vaccinia virus expression system is to deliver antigenic determinants for vaccination purposes. Progress in further attenuating vaccinia virus should lead the way to testing the efficacy of recombinant vaccinia viruses as vaccines. Viral Vectors Maintained in Episomal State. BOVINE PAPILLOMAVIRUS. Bovine papillomavirus (BPV) is a small, circular DNA containing virus that morphologically transforms rodent cells in vitro. Since BPV cannot generate infectious virus in tissue culture, it is necessary to transduce foreign DNA contained within BPV vectors by DNA-mediated transfection. Vectors containing the entire BPV genome or a 69% (5.5 kb) subgenomic transforming fragment encompassing the early region are, in some cases, stable as multicopy (20 to 100 copies/cell), extrachromosomal elements in murine NIH/3T3 and C127 fibroblast cells selected for morphological transformation. 13,~4 In other cases, the vector sequences are maintained as multiple copies integrated into the host chromosome in a head-to-tail tandem array. The multicopy nature of the DNA in BPVtransformed cells is partly responsible for the high-level expression of foreign genes contained in BPV-based vectors. Derivatives of BPV contain selectable markers encoding, for example, neomycin resistance 15 or metallothionein for heavy metal ion resistance, 16,17that obviate relying on morphological transformation to obtain cells harboring exogenous DNA (for review, see Ref. 18). One vector series, pBMT2X, contains both the human and mouse metallothionein (MT) genes as dominant selectable and amplifiable genetic markers for introduction of the BPV recombinant into cellsJ 9 Cells harboring the MT gene overexpress metallothionein, which binds excess cadmium and protects the cells against metal toxicity (up to 100/~M). Growth in increasing concentrations of cadmium can select for ,3 K. Zinn, P. Mellon, M. Ptashne, and T. Maniatis, Proc. Natl. Acad. Sci. U.S.A. 79, 4897 (1982). ~4D. DiMaio, V. Corbi, E. Sibley, and T. Maniatis, Mol. Cell. Biol. 4, 340 (1984). ~5M.-F. Law, D. R. Lowy, I. Dvoretsky, and P. Howley, Proc. Natl. Acad. Sci. U.S.A. 78, 2727 (1981). ~6M. Karin, G. Cathala, and M. C. Nguyen-Huu, Proc. Natl. Acad. Sci. U.S.A. 82, 689 (1983). ~7G. N. Pavlakis and D. H. Hamer, Proc. Natl. Acad. Sci. U.S.A. 80, 397 (1983). ~8D. DiMaio, in "The Papillomaviruses," p. 293. Plenum, New York, 1987. ~9C. M. Wright, B. K. Felber, H. Paskalis, and G. N. Pavlakis, Science 232, 988 (1986).

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cells containing an increased copy number of BPV genomes. 2° pBMT 2X has a unique XhoI cloning site into the 5' untranslated region of the mouse metallothionein I gene. Although there has been considerable success with BPV expression systems, the biology of BPV mRNA expression and the regulation of its plasmid maintenance and copy number are poorly understood. In addition, BPV vectors are prone to rearrangement. Because of this, results have been variable with different DNA inserts. As more features of the biology of BPV become understood, BPV may become a more useful system tbr expression of a wide variety of heterologous genes. Given the desirability of an episomal vector for mammalian cells and that BPV has significant disadvantages as described above, alternative vector systems have been explored. One potential system at this time is based on Epstein-Barr virus (EBV). EPSTEIN-BARR VIRUS. EBV is a herpes virus which has a large DNA double-stranded genome of 135 kb. EBV infects human B lymphocytes in vivo and in vitro. The virus normally replicates as a plasmid in infected cells. The origin of replication of the plasmid has been identified (oriP) and plasmids containing oriP can be stably maintained in the presence of the Epstein-Barr nuclear antigen (EBNA) in a variety of human, monkey, and dog cell lines, but not in rodent cell lines.2~,22 The copy number ranges from one to several hundred per cell. The mechanism which controls plasmid copy number is not known. In the lyric cycle, the genome is amplified 500- to 1000-fold by the action of the viral protein BZLF-l at a second origin of replication (oriLyt). 23 Vectors are being constructed which contain both origins for the ability to maintain plasmids within cells and then to amplify as desired by regulated expression of BZLF-1.23 A cDNA mammalian cell expression cloning vector has been constructed based on the EBV vector system for efficient transformation and expression in human cells. For this approach, cDNAs are directly cloned into an EBV expression plasmid EBO-pcD-X. 24 This plasmid contains the 2o G. N. Pavlakis, B. K. Felber, C. M. Wright, J. Papamatheakis, and A. Tse, in "Current Communications in Molecular Biology: Gene Transfer Vectors for Mammalian Cells" (J. H. Miller and M. P. Calos, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1987. 21 j. L. Yates, N. Warren, and B. Sugden, Nature (London) 313, 811 0985). 22 j. L. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. U.S.A. 81, 3806 0984). 23 W. Hammerschmidt, B. Sugden, in "Current Communications in Molecular Biology, Viral Vectors" (Y. Gluzman and S. H. Hughes, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988. 24 R. F. Margolskee, P. Kavatha, and P. Berg, Mol. Cell. Biol. 8, 2837 (1988).

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eDNA expression vector pcD-cDNA25 within an EBV replication vector. DNA from the library is introduced by electroporation into recipient cell lines. Several million transformants are selected for hygromycin resistance encoded by the vector. The transformants are screened for expression of the desired gene product. Cells expressing the product are isolated and EBO-pcD episomes are rescued by the Hirt DNA extraction procedure. The isolated DNA is used to transform competent Escherichia coli to ampicillin resistance. The EBO-pcD plasmids are recovered from the bacterial colonies and are screened for expression of the desired product after reintroduction into mammalian cells. Retroviral Vectors. Retroviruses are able to transfer their genetic information at high efficiency into eukaryotic cells. Because these enveloped RNA-containing viruses undergo a DNA stage in their replication cycle, they can be genetically manipulated to replace their own genes with exogenous genes. Since the DNA intermediate of the retrovirus can integrate into the host chromosome, these viruses can be utilized as vectors for gene transfer and insertion into the host chromosome. The development of cell lines which produce the proteins required for virus production, the reverse transcriptase (POL), the group-specific antigen (GAG), and the envelope (ENV) proteins enables the production of helper-free replication-defective recombinant viruses. Improvements in viral packaging lines have included modifications to increase the limited host range26,27and to avoid recombination leading to helper virus production.2s Presently, the most useful cell line for recombinant retrovirus production is the PA317 amphotropic packaging cell line. 2s Two vectors which have demonstrated high titers and efficient expression29 are the LNC vector, which expresses a selectable neomycin resistance gene and utilizes the CMV promoter for heterologous gene expression, and the LSN-2 vector, which utilizes the long terminal repeat for expression of foreign genes and the SV40 promoter for expression of neomycin resistance. Significant advantages of retroviral vectors include their abilities: (1) to transduce genes into a variety of cell types and into a variety of species; (2) to produce stable cell lines as a result of retrovirus integration into the host chromosome; (3) to introduce DNA into nearly 100% of the host cells, due to the high infectivity and ability to produce high-titer virus stocks; and (4) to introduce foreign genes into animals. Expression of genes introduced by retroviral infection is usually more efficient than expression from genes 25 H. Okayama and P. Berg, Mol. Cell. Biol. 3, 280 (1983). 26 R. D. Cone and R. C. Mulligan, Proc. Natl. Acad. Sci. U.S.A. 81, 6349 (1984). 2~ A. D. Miller, M. F. Law, and I. M. Verma, Mol. Cell. Biol. 5, 431 (1985). 28 A. D. Miller and C. Buttimore, Mol. Cell. Biol. 6, 2895 (1986). 29 W. R. A. Osborne and A. D. Miller, Proc. Natl. Acad. Sci. U.S.A. 85, 6851 (1988).

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introduced by DNA transfection) ° This may result from the selective retroviral integration into transcriptionally active loci or, alternatively, retroviral integration may result in transcriptional activation. However, in general, protein expression from retroviral-based vectors has been low due to problems with RNA splicing and mRNA translation. Since it is unknown which viral DNA sequences are essential for efficient expression in retroviral-based vectors, and since the insertion of different DNA sequences may impair propagation or expression of the recombinant retrovirus, success with these vectors has been variable. Furthermore, packaging constraints limit the size of the inserted segment to approximately 6 - 7 kb.

Direct DNA Transfer into Cells With most of the methods of DNA transfer, 5 - 50% of the cells in the population acquire DNA and express it transiently over a period of several days to several weeks. Eventually, the DNA is lost from the population. This transient expression of transfected DNA is conveniently used instead of the more laborious procedure of isolating and characterizing stably transfected cell lines. Transient expression experiments obviate the effects of integration sites on expression and the possibility of selecting cells which harbor mutations in the transfected DNA. Transient expression offers a convenient means by which to compare different vectors and identify that an expression plasmid is functional before establishing stable cell lines. The efficiency of expression from transient transfection is dependent on the number of cells which take up the transfected DNA, the gene copy number, and the expression level per gene. The cell line most frequently used for transient expression is derived from African green monkey kidney cells by transformation with an origin-defective mutant of simian virus 40 ( S V 4 0 ) . TM These COS cells express high levels of the SV40 large tumor (T) antigen, which is required to initiate viral DNA replication. The T antigen-mediated replication can amplify the plasmid copy number to greater than 10,000 per cell. This large copy number results in high expression levels from the transfected DNA. Vectors for Expression of cDNA Genes

Vector Components A large variety of transient expression vectors have been described recently. In many cases, it is difficult to evaluate results from different vectors used in different laboratories. Most expression vectors are designed 3o L. H. S. Hwang and E. Gilboa, J. Virol. 50, 417 (1984). 3~ p. Mellon, V. Parker, Y. Gluzman, and T. Maniatis, Cell 27, 279 (1981).

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to accommodate eDNA rather than genomic clones, eDNA clones are more convenient to manipulate due to their smaller size. Today, most useful vectors contain multiple elements which include (l) an SV40 origin of replication for amplification to high copy number in COS monkey cells, (2) an efficient promoter element for transcription initiation, (3) mRNA processing signals which include mRNA cleavage and polyadenylation sequences and frequently also intervening sequences, (4) polylinkers that contain multiple endonuclease restriction sites for insertion of foreign DNA, and (5) selectable markers that can be used to select cells that have stably integrated the plasmid DNA. The level of protein expression from heterologous genes introduced into mammalian cells depends on multiple factors, including DNA copy number, efficiency of transcription, mRNA processing, transport, stability, and translational efficiency, and protein processing, secretion, and stability. The rate-limiting step for high-level expression may be different for different genes. Constitutive Promoters. Two important identified sequence elements that control transcription initiation are the promoter and the enhancer. The best-characterized promoter systems are those derived from viruses (SV40 and adenovirus), primarily due to the availability and analysis of mutations generated by natural variation of the virus (for a review, see Ref. 32). The promoter is generally composed of the TATAA box (often at approximately - 3 0 bp with respect to the mRNA initiation site as + 1),33 which probably functions by designating the start site for RNA polymerase II transcription, and the CAAT box (at approximately - 8 0 bp from the mRNA initiation site), 34 where factors bind to facilitate transcription initiation. Most vectors for mammalian cells contain promoter elements from efficient transcription elements such as the SV40 early promoter, the Rous sarcoma virus promoter, the adenovirus major late promoter, and the human cytomegalovirus (CMV) immediate early promoter. Transcriptional enhancers encompass a variety of core sequences that act to increase transcription from a promoter in an orientation- and distance-independent manner. 35 The transcriptional enhancer appears to be a primary regulator of transcriptional activity. Some enhancers located in viruses, for example, polyoma36 or Moloney murine sarcoma virusy show 32 S. McKnight and R. Tjian, Ce1146, 795 (1986). 3a R. Breathnach and P. Chambon, Annu. Rev. Biochem. 50, 349 (1981). C. Benoist, K. O'Hare, R. Breathnach, and P. Chambon, Nucleic Acids Res. 8, 127 (1980). 35 E. Serfling, M. Jasin, and W. Schaffner, Trends Genet. 1, 224 (1985). J. deVilliers, L. Olson, J. Banerji, and W. Schaffner, Cold Spring Harbor Syrup. Quant. Biol. 47, 911 (1983). 37 L. A. Laimins, G. Khoury, C. Gorman, B. Howard, and P. Gruss, Proc. Natl. Acad. Sci. U.S.A. 79, 6453 (1982).

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a host cell preference and, thus, contribute to the host range of the virus. Others, like the SV40 enhancer, as the Rous sarcoma virus (RSV) long terminal repeat (LTR), a9 and the human CMV enhancer,4° are very active in a wide variety of cell types from many species. Enhancers with strict cell-type specificity have been observed in many cellular genes which exhibit tissue specificity of expression, for example, the immunoglobulin genes 41-43 and the insulin gene.~ Enhancers are likely the primary cisacting determinants in tissue specificity of gene transcription. The addition of a strong enhancer can increase transcriptional activity by 10- to 100fold. Thus, most expression vectors include a strong enhancer, frequently derived from SV40, RSV, or CMV. Inducible Promoters. In order to express a protein which is potentially cytotoxic, it is advisable to use an inducible expression system which is regulated by an external stimulus. Sequences required for induced transcription from a number of promoters have been identified and function as enhancer elements. In selecting an inducible vector system for a particular gene, it is important to ensure that the inducing stimulus does not interfere with properties under study. It is also important to know what fold induction is desirable and the maximal achievable expression level. In many cases, the fold induction may be large but the maximal level of expression is low compared to a constitutive promoter. Alternatively, the fold induction may be low but the maximal expression level very high. Several of the inducible promoters which have demonstrated utility for expression of heterologous genes are described below. INTERFERON fl PROMOTER. The interferon fl gene is highly inducible in fibroblasts by virus infection or by the presence of double-stranded RNA [poly(rI)-poly(rC)] (for review, see Ref. 45). The sequences responsible for its induction are found between bases from - 77 to - 36 from the start site for mRNA transcription. This system has been used to obtain a 200-fold induction of interferon fl expression in Chinese hamster ovary cells after cointroduction and coamplification of interferon fl genes with a dihydrofolate reductase (DHFR) gene.~ These experiments demonstrated that the as G. Neuhaus, G. Neuhaus-Uri, P. Gruss, and H.-G. Schweiger, EMBO J. 3, 2169 (1984). 39 C. M. Gorman, G. D. Merlino, M. C. Willingham, I. Pastan, and B. H. Howard, Proc. Natl. Acad. Sci. U.S.A. 79, 6777 (1982). 40 M. Boshart, F. Weber, J. Gerhard, K. Dorsch-Hasler, B. Fleckenstein, and W. Schaffner, Cell41, 521 (1985). 41 j. Banerji, L. Olson, and W. Schaffner, Cell33, 729 (1983). 42 S. D. Gillies, S. L. Morrison, V. T. Oi, and S. Tonegawa, Cell33, 717 (1983). 43 C. Queen and D. Baltimore, Cell33, 741 (1983). 44 H. Ohlsson, and T. Edlund, Cell45, 35 (1986). 45 p. Lengyel, Annu. Rev. Biochem. 51, 251 (1986). 46 F. McCormick, M. Trahey, M. Innis, B. Dieckmann, and G. Ringold, Mol. Cell. Biol. 4, 166 (1984).

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normal inducibility of the interferon fl gene is maintained in cells containing 25-fold amplified copies of the interferon gene. One potential problem with this induction system is that induction of murine fibroblasts results in a response in only 5-10% of the cells in the population. 47 These same studies demonstrate poor activation of the interferon fl gene in human HeLa cells, but that pretreatment with interferon results in high inducibility. Thus, an interferon-inducible factor appears to be required for activation of the interferon fl gene in the nonresponsive cells. HEAT-SHOCK PROMOTER. Heat-shock genes are transcriptionally activated when cells are exposed to hyperthermia or a variety of other stresses? s The molecular basis for this gene activation has been extensively studied using the Drosophila heat-shock protein 70 gene (hsp70) promoter, which is efficiently expressed and tightly regulated in Drosophila and mammalian cells. The Drosophila hsp70 promoter contains a 15-bp sequence upstream from the TATA box which is responsible for activation of the promoter in response to heat and which can activate a heterologous thymidine kinase (TK) gene when placed upstream from the TK TATA box. 49 This sequence is the binding site for a specific heat-shock transcription factor that is activated during heat shock. 5°,5~ This inducible system has been used to obtain high level of expression of the c-myc protein in CHO cells after coamplification of a hsp70 promoter-c-myc gene fusion with DHFR. 52 Highly amplified cell lines were obtained that contain 2000 copies of the introduced c-myc fusion gene, and undetectable levels of c-myc mRNA. Incubation of these cells at 43 ° resulted in at least a 100-fold induction ofc-myc mRNA. Translation only occurred when the cells were returned to 37 °. After 3 - 4 hr, the c-myc protein levels reached approximately 1 mg/109 cells. These results demonstrate that, even at a level of 2000 copies, the hsp70 promoter retains inducibility. This suggests that any negative or positive acting factor involved in hsp70 promoter activity is not limiting. One potential problem with this system is that heat shock is detrimental to the cells and may severely affect the protein secretion machinery of some cell lines. 53 METALLOTHIONEIN PROMOTER. Metallothioneins are small cysteinerich proteins which play an important role in detoxification of heavy 47 T. Enoch, K. Zinn, and T. Maniatis, Mol. Cell. Biol. 6, 801 (1986). 48 H. R. B. Pelham, Trends Genet. 1, 31 (1985). 49 H. R. B. Pelham and M. Bienz, E M B O J . 1, 1473 (1982). 5o C. Wu, Nature (London) 311, 81 (1984). 51 C. S. Parker and J. Topoi, Cell37, 273 (1984). s2 F. M. Wurm, K. A. Gwinn, and R. E. Kingston, Proc. Natl. Acad. Sci. U.S.A. 83, 5414 (1986). ~3 M. J. Schlesinger, J. Cell Biol. 103, 321 (1986).

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VECTORS USED FOR EXPRESSION

499

metals and in heavy metal hemostasis. 54 Metallothionein (MT) gene transcription is induced by the presence of heavy bivalent metal ions, such as cadmium and zinc, 55 by glucocorticoid hormones, 56 and by interferonY There is a cellular factor(s) that, in the presence of cadmium, interacts with the heavy metal-responsive element to induce transcription. 5s The mouse MT-I promoter region contains a metal regulatory element between bases - 59 and - 4 6 , with respect to the start site for mRNA transcription as + 1, which may act as an inducible enhancer. 59,6°Insertion of multiple copies of this sequence can confer zinc "inducibility" on a heterologous promoter. 6' The mouse MT-I promoter has been used successfully in bovine papilloma-based vector systems. '7,~9 However, the basal level of expression is generally high, and the induction ratio is poor, generally never greater than 5- to 10-fold. In addition, high levels of cadmium are cytotoxic. Zinc may be used as a less toxic inducing agent. GLUCOCORTICOID INDUCTION. Glucocorticoids and other steroid hormones mediate physiological responses in target cells via a specific, highaffinity interaction with cytoplasmic receptors. Hormone binding to receptors results in a conformational change, thereby allowing the hormone-receptor complex to bind specific sites on DNA. 62 H o r m o n e receptor complex binding to DNA results in transcriptional activation of the hormone-responsive gene. The best studied glucocorticoid-inducible promoter is the mouse mammary tumor virus (MMTV) LTR. The glucocorticoid-responsive element (GRE) behaves as an enhancer element and has been localized between - 1 0 0 and - 2 0 0 of the MMTV LTR. The purified glucocorticoid receptor protein binds to the GRE sequence in vitro 63,~ and has been cloned and extensively characterized.~5-67 54 p. E. Hunziker and J. H. R. Kaegi, Top. MoL Struct. Biol. 7, 5712 (1981). 55 D. M. Durnam and R. D. Palmiter, J. BioL Chem. 256, 5712 (1981). 56 L. J. Hager and R. D. Palmiter, Nature (London) 291, 340 (1981). 57 R. L. Friedman, S. P. Manly, M. McMohon, I. M. Kerr, and G. R. Stark, Cell 38, 745 (1984). 5s C. Sequin, B. K. Felber, A. D. Carter, and D. H. Hamer, Nature (London) 312, 781 (1984). 59 E. Serfling, A. Lubbe, K. Korsh-Hasler, and W. Schaffner, EMBOJ. 4, 3851 (1985). 60 M. Karin, A. Haslinger, H. Holtgreve, G. Cathala, E. Slater, and J. D. Baxter, Cell 36, 371 (1984). 6t p. F. Searle, G. W. Stuart, and R. D. Palmiter, MoL Cell. Biol. 5, 1480 (1985). 62 G. M. Ringold, Annu. Rev. PharmacoL ToxicoL 25, 529 (1985). 63 F. Payvar, D. DeFranco, G. L. Firestone, B. Edgar, O. Wrange, S. Okret, J. A. Gustaffson, and K. R. Yamamoto, Cell35, 381 (1983). H. M. Jantzen, U. Strahle, B. Gloss, F. Stewart, W. Schmid, M. Boshart, R. Miksicek, and G. Schuts, Ce1149, 29 (1987). 65 V. Giguere, S. M. Hollenberg, M. G. Rosenfeid, and R. M. Evans, Cel146, 645 (1986). 66 p. j. Godowski, S. Rusconi, R. Miesfeld, and K. R. Yamamoto, Nature (London) 325, 365 (1987). 67 S. Rusconi and K. R. Yamamoto, EMBO J. 6, 1309 (1987).

500

EXPRESSION IN MAMMALIAN CELLS

supF

M13ori ~ * * ~ A ~ M V f l - 7

,VXori I I ~k SV40ori ~

C.[)." 8 H.4 kl:))

~

promoter

~st'/J- stuffer ~,~BstXl ~ estI / / NOtI ~ / ..

[39]

SV40 ~'--,-.~rVenh AmpR/ ~dMLP // \ ~ k TPL '

pMT2

~Sst !

kb)

ori :..~

'EcoRI

//DHFR

spl~e+An

Pyori

VA 1

Poly(A)

FIG. 1. Efficient cloning-expression vectors CDM8 [B. Seed and A. Arufro, Proc. Natl. Acad. Sci. U.S.A. 84, 3365 (1987)] and pMT2 [R. J. Kaufman, M. V. Davies, V. Pathak, and J. W. B. Hershey, Mol. Cell. Biol. 9, 946 (1989)] for mammalian cells.

A conditional glucocorticoid-inducible expression vector has been developed for high-level inducible expression in Chinese hamster ovary cells. 68 This expression system requires high-level expression of the glucocorticoid receptor protein in the host cell and multiple copies of the glucocorticoid responsive element within the expression vector. To accomplish this, the rat glucocorticoid receptor was expressed at high level in DHFR-deficient Chinese hamster ovary cells (called GRA cells.) 68 When multiple copies of the glucocorticoid-responsive element from MMTV are inserted into the expression vector (pMT2; see Fig. 1) to yield pMG 18PC, addition of dexamethasone yields a 7-fold induction of expression over the high constitutive level (Fig. 2). When the constitutive SV40 enhancer is delected from pMG18PC to yield pMG18oS, basal expression is eliminated and maximal expression upon addition ofdexamethasone is retained (Fig. 2). This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion. As the genes for other transactivators are cloned and characterized for the structural features required to activate gene transcription, it will be possible to engineer novel potent transactivators to elicit induction of specific promoters. This has recently been demonstrated by mixing DNA binding and transcriptional activation domains from yeast, bacteria, and mammalian cells to elicit transactivation of specific genes. 69-7~ 6s D. I. Israel and R. J. Kaufman, Nucl. Acids Res. 17, 4589 (1989). 69N. Webster, J.-R. Jin, S. Green, M. Hollis, and P. Chambon, Cell52, 169 (1988). ToK. Lech, K. Anderson, and R. Brent, Ce1152, 179 (1988). 7t H. Kakidani and M. Ptashne, Ce1152, 161 (1988).

[39]

VECTORS USED FOR EXPRESSION Mock _

pMT2 I

pMG 18PC

_

+

2

3

II

_

+

4

5

501

pMG 18&S II

_

+

6

7

Plasmid ]

Dexamethasone

Cells: CHO

1

GRA ~-DHFR

1

2

3

4

5

6

7

FIG. 2. Glucocorticoid-induced expression in transiently transfected Chinese hamster ovary cells. Plasmid DNA was transfected by the DEAE-dextran protocol into the indicated cell lines. At 24 hr posttransfection, dexamethasone was applied to one of two parallel plates (+ or - as indicated). Total RNA was isolated 48 hr posttransfecfion and analyzed for dihydrofolate reductase (DHFR) RNA by Northern blot hybridization to a DHFR-specific probe. The glucocorticoid expression vectors pMG 18PC and pMG 18AS indicated are similar to pMT2 except for the insertion of 18 glucocorticoid-responsive elements, pMG18AS has deleted the SV40 enhancer present in pMT2. The transfected cell lines are the original Chinese hamster ovary dihydrofolate reductase-deficient cells (CHO) [L. A. Chasin and G. Urlaub, Proc. Natl. Acad. Sci. U.S.A. 77, 4216 0980)] and the same cells (GRA) which express high levels of the rat glucocorticoid receptor. For details, see Ref. 68.

RNA Processing. SPLICING. Most higher eukaryotic genes contain introns which are processed from the precursor RNA in the nucleus to generate the mature mRNA. The sequences and factors responsible for RNA splicing have recently been reviewed. 72 Although many genes do not require introns for mRNA formation when introduced into mammalian cells, there are several examples of genes that have strong requirements for the presence of an intron. ~3,74 In general, it is probably better to include introns in cDNA expression vectors for mammalian cells. For example, the intervening sequences from SV40 small t antigen mRNA or hybrid intron composed from adenovirus and immunoglobulin sequences are frequently used (see Fig. 2). In contrast to expression in mammalian cell lines, there is 72 p. A. Sharp, Science 235, 766 (1987). 73 A. R. Buchman and P. Berg, Mol. Cell. Biol. 8, 4395 0988). 74 B. Gruss and G. Khoury, Nature (London) 286, 634 (1980).

502

EXPRESSION IN MAMMALIAN CELLS

[39]

a profound requirement for the presence of introns for expression of genes introduced into transgenic animals. 75 TRANSCRIPTION T E R M I N A T I O N AND POLYADENYLATION. T r a n s c r i p t i o n termination, 3' end cleavage, and polyadenylation of precursor

mRNAs are essential steps for the biogenesis of mRNA (for review, see Ref. 76). In higher eukaryotes, RNA polymerase II transcribes across the polyadenylation site(s) and terminates downstream of the mature 3' termini for the mRNA. To date, the DNA sequences which function as transcription termination signals have only been mapped to various restriction fragments at the 3' end of several g e n e s . 77-79 Structural features within some of these regions have been compared to identify a sequence which represents a conserved element within the termination region of a number of different genes. 8° Further studies are required to identify whether this sequence has any functional significance. The possibility that an upstream promoter may occlude transcription from a downstream promoter, due to polymerase transcribing through the downstream promoter, s~ suggests that insertion of transcription termination signals upstream from the second transcription unit in an expression vector may potentiate expression from that promoter by preventing transcription from extending through the upstream sequences. Indeed, this has been directly demonstrated by the insertion of a histone H2A gene termination region or a mouse fl-globin gene termination region between two tandem a-globin transcription units to elicit an approximately 7-fold increase in expression from the downstream transcription unit. s2 Transcription termination signals would also provide an approach to minimize transcription from the opposite strand of DNA, which can suppress gene expression through the formation of antisense mRNA. s3,s4 The 3' end of eukaryotic mRNA is formed by polyadenylation, which involves cleavage of the precursor mRNA at a specific site and then polymerization of about 200 adenylate residues, poly(A), to the newly 75 R. Brinster, J. Allen, R. Behringer, R. Gelinas, and R. Palmiter, Proc. Natl. Acad. Sci. U.S.A. 85, 836 (1988). 76 M. L. Birnstiel, M. Susslinger, and K. Strub, Cell41, 349 (1985). 77 E. G. Frayne, E. J. Leys, G. F. Crouse, A. G. Hook, and R. E. Kellerns, Mol. Cell. BioL 4, 2921 (1984). 7s E. Falck-Pederson, J. Logan, T. Shenk, and J. E. Darnell, Jr., Cell40, 897 (1985). 79 C. Birchmeier, D. Schumperli, G. Sconzo, and M. L. Birnstiel, Proc. NatL Acad. Sci. U.S.A. 81, 1057 (1984). so E. G. Frayne and R. E. Kellems, Nucleic Acids Res. 14, 4113 (1986). st B. R. Cullen, P. T. Lomedico, and G. Ju, Nature (London) 307, 241 (1984). s2 N. J. Proudfoot, Nature (London) 322, 562 (1986). s3 j. G. Izant and H. Weintraub, Science 229, 345 (1985). D. A. Melton, Proc. NatL Acad. Sci. U.S.A. 82, 144 (1985).

[39]

VECTORS USED FOR EXPRESSION

503

generated 3' end. s5 Removal of the polyadenylation site decreases expression up to 10-fold. s6 Two sequences important for polyadenylation have been identified. The first is a highly conserved hexanucleotide, AAUAAA, that is present 11 - 30 nucleotides upstream of most polyadenylation sites, and which forms the recognition sequence for the cleavage and polyadenylation reaction. Deletions or point mutations in this sequence prevent the appearance of properly polyadenylated mRNA in vivo. 87-89 There is also a requirement for a sequence downstream of the poly(A) site for efficient cleavage and polyadenylation. 9°-92 A loose consensus sequence, for this potential second element, has been identified as either a U-rich or a (G + U)-rich tract. 9a However, removal of this sequence, in some instances, has no effect on the efficiency of polyadenylation 94 but may influence the position of 3' processing. 95 This sequence appears to be required for formation of a precleavage complex. 96 Efficient signals for polyadenylation from SV40 early transcription unit, the hepatitis B surface antigen transcription unit, and the mouse fl-globin gene are commonly used. Translation of mRNAs. The primary event limiting translation of mRNAs is at the initiation step (for a review, see Ref. 97). Polypeptide chain synthesis is initiated when a ternary complex of eukaryotic initiation factor 2, GTP, and initiator Met-tRNA binds a 40S ribosomal subunit. This 40S ribosomal subunit complex binds to the 5' end of the mRNA and migrates in the 3' direction until it encounters an A U G triplet which can efficiently serve as the initiator codon. Subsequently, a 60S ribosomal subunit joins to form an 80S initiation complex. Several approaches to develop expression vectors optimized for protein synthesis initiation are discussed below. 8s C. L. Moore and P. A. Sharp, Cell41, 845 (1985). 86 R. J. Kaufman and P. A. Sharp, Mol. Cell. Biol. 2, 1304 (1982). 87 C. Montell, E. F. Fisher, M. H. Caruthers, and A. J. Berk, Nature (London) 305, 600 (1983). s 8 D. R. Higgs, S. E. Y. Goodbourn, J. Lamb, J. B. Clegg, D. J. Weatherall, and N. J. Proudfoot, Nature (London) 306, 398 (1983). s9 M. Fitzgerald and T. Schenk, Cell 24, 251 (1981). 90 M. Wickens and P. Stephenson, Science 226, 1045 (1984). 9t M. A. McDevitt, M. J. Imperiale, H. Ali, and J. R. Nevins, Cel137, 993 (1984). 92 M. Sasofsky, S. Connelly, J. L. Manley, and J. C. Alwine, Mol. Cell. Biol. 5, 2713 (1985). 93 A. Gil and N. Proudfoot, Cell49, 399 (1987). 94 D. Danner and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 82, 8658 (1985). 9s p. j. Mason, J. A. Elkington, L. M. Maigorazata, M. B. Jones, and J. G. Williams, Cell 46, 263 (1986). 96 D. Zarkower and M. Wickens, J. Biol. Chem. 263, 5780 (1988). 97 V. Pain, Biochem. J. 235, 625 (1986).

504

EXPRESSION IN M A M M A L I A N CELLS

[39]

MRNA AND CONSENSUS SEQUENCE REQUIREMENTS. The sequence requirements for most efficient m R N A translation initiation are the following: 9s 5'CC~CCATGG - 3'. Of most importance is the purine in the - 3 position and then the G in the + 4 position. Mutations to alter this consensus sequence around the initiator methionine codon may reduce translation as much as 10-fold. In addition to the consensus sequence requirements, there are other features that influence m R N A translation. The presence of upstream initiation codons is detrimental to initiation at the downstream site, particularly if they are not followed by in-frame termination codons before the start site for translation initiation of the desired coding region. 99,1°° In addition, the presence of significant secondary structure ( - 5 0 kcal/mol) near the A U G on the m R N A can be detrimental to translation. 1Ol-io3 Most vectors are designed to contain 5' untranslated sequences derived from the particular promoter element used for transcription initiation. TRANSLATIONAL EFFICIENCY MEDIATED BY EIF-2. Translation of m R N A derived from plasmid DNA after transient transfection of COS-1 cells may be inefficient. This deficiency can be corrected by the introduction of the adenovirus virus-associated (VA) RNA gene into the COS-1 cell expression vector.l°4 VA RNA is a small RNA synthesized by RNA polymerase III which has been proposed to block the double-stranded RNAactivated (DAI) protein kinase. 1°5,1°~ It has been proposed that transcription from both strands of the episomal DNA template in transfected COS-l cells can generate double-stranded RNA, leading to activation of DAI kinase and translation arrest. VA RNA stimulates translation by preventing the phosphorylation of the a subunit of eIF-2 a. 1°4,1°7,1°s A similar stimulation in translation results from expression of serine to alanine mutant of eIF-2, which renders it phosphorylation resistant. 1°9 To date, there has been no demonstration of an effect of VA RNA on the 9s M. Kozak, Cell44, 283 (1986). 99 C.-C. Liu, C. C. Simonsen, and A. D. Levinson, Nature (London) 309, 82 (1984). i o o M. Kozak, Nucleic Acids Res. 12, 3873 (1984). 1or j. Pelletier and N. Sonenberg, Cell 40, 515 (1985). 1o2 M. Kozak, Proc. Natl. Acad. Sci. U.S.A. 83, 2850 (1986). io3 j. Pelletier and N. Sonenberg, Biochem. Cell Biol. 65, 576 (1987). 1o4R. J. Kaufman, Proc. Natl. Acad. Sci. U.S.A. 82, 689 (1985). 1o5 j. Kitajewski, R. J. Schneider, B. Safer, S. M. Munemitsu, C. E. Samuel, B. Thimmappaya, and T. Schenk, Cel145, 195 (1986). io6 R. P. O'Malley, T. M. Mariano, J. Siekierka, and M. B. Mathews, Cell44, 391 (1986). 1o7C. Svensson and G. Akusjarvi, E M B O J . 4, 957 (1985). los R. J. Kaufman and P. Murtha, Mol. Cell. Biol. 7, 1568 (1987). ,o9 R. J. Kaufman, M. V. Davies, V. Pathak, and J. W. B. Hershey, Mol. Cell. Biol. 9, 946 (1989).

[39]

VECTORS USED FOR EXPRESSION

505

translation of m R N A derived from transcription units integrated in the host chromosome. POLYClSTRONIC EXPRESSION VECTORS. It is now known that polycistronic mRNAs are translated in mammalian cells) ~° One mechanism which has been postulated is that translation termination occurs and ribosome scanning continues with reinitiation at downstream A U G s ) ~t However, there are at least some viral mRNAs of the picornavirus family that contain specific sequences to promote internal ribosome binding and translation initiation. ~2,~t3 The utilization of polycistronic mRNA expression vectors containing the desired gene in the 5' open reading frame and a selectable gene in the 3' position has been used to select cells that express high levels of the gene product in the 5' position.tta Since the translation of the selectable gene in the 3' end is inefficient, polycistronic expression vectors exhibit preferential translation of the 5' desired gene and cells require high levels of polycistronic mRNA to survive selection. Preparation ofcDNA Inserts. The most common approach to express a foreign gene relies on insertion of a cDNA copy encoding the desired protein into an appropriate expression plasmid. Generally, cDNA copies are used, since they are easier to manipulate in vitro due to their smaller size. In addition, the appropriate coding region can be inserted behind an efficient promoter which also includes all expression elements to ensure efficient expression. In preparation of a cDNA insert for introduction into any particular vector, a couple of suggestions may improve expression. First, if the cDNA were obtained by addition of homopolymeric tails into the 5' noncoding region, they should be removed. Removal of GC tails may improve expression up to 10-fold) ~5 Second, if the AUG does not conform to the consensus GCCAUGG, it is recommended that the context of the initiation codon should be changed so that it does conform. Third, in general and when convenient, it is better to remove as much as possible of the 5' and 3' untranslated regions of the cDNA. This is particularly true if the cDNA contains AUG initiation codons which are upstream of the authentic AUG or if the 5' untranslated region contains extensive secondary structure. 99- io3 In addition, sequences within the 3' end of the mRNA may reduce mRNA stability.tt6

Ho D. Peabody and P. Berg, Mol. Cell. BioL 6, 2695 (1986). ttl M. Kozak, Cell47, 481 (1986). .2 j. Pelletier and N. Sonenberg, Nature (London) 334, 320 (1988). It3 S. K. Jang, M. V. Davies, R. J. Kaufman, and E. Wimmer, J. Virol. 63, 1651 (1989). ,4 R. J. Kaufman, P. Murtha, and M. Davies, EMBOJ. 6, 187 (1987). ,5 C. C. Simonsen et aL, ICN-UCLA Symp. Mol. Cell. Biol. 25, 1 (1981). tit G. Shaw and R. Kamen, Ce1146, 659 (1986).

506

EXPRESSION IN MAMMALIAN CELLS

[39]

Efficient Vectors for Expression of cDNAs in Mammalian Cells Two vectors which have successfully been used in different laboratories are shown in Fig. 1. CDM8 t~7 contains a human cytomegalovirus (CMV) promoter for efficient transcription in a variety of cell types in vivo and a promoter from T7 bacteriophage DNA-dependent RNA polymerase for generation of transcripts in vitro. It contains SV40 and polyoma origins of replication for replication in COS monkey cells and polyoma virus-transformed mouse fibroblasts. Transcription is driven by the human CMV promoter and the vector contains an SV40 intervening sequence and polyadenylation signal. CDM8 contains a n VX origin of replication for propagation in E. coli and a suppressor tRNA gene (supF) for selection in E. coli. These elements permit the plasmid to be conveniently isolated from mammalian cells through isolation of episomal DNA by the Hirt procedure~~8and used to transform E. coli strain MCI061 (p3) to tetracycline and ampicillin resistance by suppression of amber mutations in the tetracycline resistance gene and p-lactamase genes carried on the p3 plasmid. An alternative method to isolate the plasmid DNA from mammalian cells involves cloning DNA into amber mutant strains of bacteriophage 2 and selecting phage by complementation mediated by supF. CDM8 also contains an M 13 origin of replication which facilitates oligonucleotide-directed mutagenesis using single-stranded DNA templates, and it has two BstNI restriction endonuclease sites for cloning cDNAs into the vector at high efficiency in the proper orientation by the use of oligonucleotide adapter sequences. pMT21°9 contains plasmid sequences from puc 18 which allow for propagation and selection for ampicillin resistance in E. coli. It contains the SV40 origin of replication and enhancer element. It utilizes the adenovirus major late promoter for transcription initiation. Contained within the 5' end of the mRNA is the tripartite leader from adenovirus late mRNA and a small intervening sequence. There are several cloning sites, including EcoRI and PstI, for insertion of foreign DNA. In the 3' end of the transcript there is a cleavage and polyadenylation signal from the SV40 early region. In addition, this plasmid contains the adenovirus VAI gene, which is a polymerase III transcription unit which encodes a small RNA that potentiates translation of the plasmid-derived mRNA in transfected COS monkey cells. This vector also contains a DHFR coding region in the 3' end of the transcript. Expression of DHFR from resulting bicistronic mRNAs can be used to select directly for DHFR expression in Chinese ~ B. Seed and A. Aruffo, Proc. Natl. Acad. Sci. U.S.A. 84, 3365 (1987). lib B. J. Hirt, J. Mal. Biol. 26, 365 (1967).

[39]

VECTORS USED FOR EXPRESSION

507

hamster ovary cells which are DHFR d e f i c i e n t . 114,119 In addition, it is possible to select directly for amplification of the copy number by selection for resistance to increasing concentrations of methotrexate, an inhibitor of DHFR. Transient Expression of Plasmid DNA in COS-1 Cells The most widely used and convenient system for expression of a foreign gene is by introduction of DNA into COS-1 cells and then monitoring expression over the next 48- 72 hr. Although there are many vectors suitable for this purpose, a very efficient vector is pMT2 (see Fig. 1). The following is a protocol that can be used to obtain efficient expression by this approach.

DEAE-Dextran-Mediated Transfection of COS-1 Cells ~2° Stock Solutions (10 ×) DEAE-Dextran (Pharmacia, MW 500,000): 2.5 mg/ml in Dulbecco's modified essential (DME) medium and stored at 4* (10×) 1 M Tris-HC1, pH 7.3: stored at 4* (1000 ×) Chloroquine (Sigma): 0.1 M stored at - 20 ° in dark (1 ×) 10% DMSO reagent (1 liter): 137 m M NaC1 (8 g), 5 m M KCI (0.37 g), 0.7 m M NH2HPO4 (0.01 g), 6 m M D-glucose (1.08 g), 21 m M HEPES (5 g). Then add 900 ml H20 to take pH to 7.1 and filter through a 0.2/zm filter. Finally, add 100 ml 100% DMSO

Cells. COS- 1 cells are grown in DME medium supplemented with 10% heat-inactivated fetal calf serum. They are usually subcultured twice per week at a 1 : 4 or 1 : 6 split ratio. Growth Medium. Dulbecco's modified essential medium with 2 m M glutamine, 100 U/ml streptomycin, 100/zg/ml penicillin, and 10% heat-inactivated fetal calf serum. Transfection 1. Subculture cells 1 : 6 into 100 m M tissue culture plates at 12- 24 hr before transfection. 2. Aspirate medium and wash 2 times with 7 ml each of serum-free DME. Note that the cells will die if the DEAE-dextran and the serum contact the cells at the same time. 3. Feed the cells the D N A - m e d i u m mix (4 ml per 100-ram culture 119L. A. Chasin and G. Urlaub, Proc. NatL Acad. Sci. U.S.A. 77, 4216 (1980). 120j. H. McCutchan and J. S. Pagano, JNCI, J. NatL Cancerlnst. 41, 351 (1968).

508

EXPRESSION IN MAMMALIAN CELLS

[39]

dish containing 8/tg of DNA) which is prepared as follows: (a) Add DNA [generally prepared in sterile Tris-HC1 (10 raM, pH 8.0) and EDTA (l raM)] to 0.4 ml of Tris-HC1 (final DNA concentration should be 2 pg/ml in the medium). Mix well. (b) Add 0.4 ml of 10× DEAE-dextran to DNA-Tris-HC1. (c) Add 3.2 volumes of DME that contains 2 m M glutamine, 100 U/ml streptomycin, 100/zg/ml penicillin. Mix well. 4. Incubate for 8 - 10 hr at 37 °. 5. Rinse once with 7 ml of serum-free DNA. 6. Add 2 ml of 10% DMSO reagent to the dish. Leave for 2 - 3 min at room temperature. Aspirate. 7. Add 5 ml/plate o f D M E + 10% fetal calf serum and 0. l mMchloroquine for 2.5 hr. 8. Remove chloroquine and rinse once with serum-free medium and add l 0 ml of DME growth medium per plate. 9. After 30 hr, aspirate medium and feed 10 ml of fresh growth medium (optional). 10. After 4 8 - 72 hr, harvest medium and/or cells as desired.

Notes. This technique results in significant cell death. In the optimal experiment, approximately 25% of the cells will die. Of the remaining cells, 20% usually acquire and express the DNA. The toxicity is most evident when the transfected cells are less than 30% confluent at the start of the transfection. It is recommended to use CsCl-banded DNA for this procedure. However, miniplasmid DNA preparations may be used but yield significantly lower levels of expression. Experimental Approachfor Expression of Foreign Genes in Mammalian Cells Although many approaches may be taken to express a particular gene, the choice is very much dependent o n t h e requirements for the questions that are to be answered. Table I lists the advantages and expression levels obtained with a variety of different expression systems. The most convenient system and the one which can yield the most rapid results is transient expression in COS cells. Success using the COS system will also provide incentive for any more laborious approach by ensuring that the eDNA carded in the expression plasmid can direct synthesis of the desired gene product. To monitor efficient expression in COS cells, it is necessary to include a positive control in order to compare expression of the desired gene with a gene that is known to be expressed well. This comparison rules out potential problems that may arise from the transfection. It will ensure that the transfection efficiency is adequate and that the COS cells are appropriate.

[39]

VECTORS USED FOR EXPRESSION

509

TABLE I EXPRESSION LEVELS AND UTILITIES FOR DIFFERENT MAMMALIAN CELL EXPRESSION SYSTEMS

Cell line

Mode of DNA transfer

Monkey cells CV-1

SV40 virus infection

Optimal expression level (ag/ml)

Primary utility

1 - 10

Expression of wild-type and mutant proteins Cloning by expression in mammalian cells; rapid characterization of cDNA clones; expression of mutant proteins

COS

Transient DEAE-dextran DNA transfection

1

CV- 1

Transient DEAE-dextran DNA transfection

0.05

BPV stable transformant

l- 5

Murine-fibroblasts C 127 3T3

Retrovirus infection

CHO-DHFR-

Stable DHFR + transformant Amplified MTX r

Primate/rodent

0.1-0.5

High-level constitutive expression Gene transfer into animals; expression in different cell types

0.01 - 0.05

Vaccinia virus EBV vector

10 1 NA

High-level constitutive expression Vaccines Cloning by expression

aNA, Not available.

In addition, this comparison may also provide insight as to why a particular gene may not be efficiently expressed. As an example of this comparison, Fig. 3 shows the results of transfected COS cells labeled with [asS]methionine for 20 min at 48 hr posttransfection and then extracted by lysis in RIPA ~2~buffer and electrophoresis on SDS-polyacrylamide gels. Cells transfected with the control DHFR expression plasmid pMT2 show a major product at 20 kDa which represents DHFR synthesis. When a heterologous coding region for human adenosine deaminase (ADA) is inserted into the cloning site to the 5' end of the DHFR-coding region, the transfected cells show a major band which represents ADA expression at approximately 44 kDa. The efficiency of ADA expression is roughly similar to that obtained for DHFR. Thus, we conclude that the expression plasmid is working appropriately for COS cells. Expression of an efficiently expressed secreted protein is shown in 121 A. J. Dorner and R. J. Kaufman, this volume [44].

510

[39]

EXPRESSION IN MAMMALIAN CELLS

Cell Extract

Cond. Medium

< a o

t-

t-

o

O9

~- C S F

A c t i n _~ A D A -* !!!~i!i i i!! ! i i i i!i!!i !!!!ii ~ ii!iiiiiiiiiiiiiiiill~iiiiiiiiiiiiiiii~

DHFR-) 1

2

3

4

5

Fio. 3. Analysis of transiently transfected COS-I monkey cells. The pMT2 expression plasmids encoding DHFR (MT2), human adenosine deaminase (ADA, MT2ADA), and a colony-stimulating factor (CSF) were transfected into COS-I monkey kidney cells. At 48 hr posttransfection cells were pulse-labeled with [35S]methionine for 20 min (cell extracts) or 3 hr (conditioned medium), and samples of the total cell extracts or conditioned medium were prepared for SDS-polyacrylamide gel electrophoresis. The results show the expression obtained for intracellular (ADA and DHFR) and secreted (CSF) proteins.

[39]

VECTORS USED FOR EXPRESSION

51 1

lane 5. In this case, transfected cells are labeled for 4 hr in order to permit secretion of the expressed protein and the labeled conditioned medium taken directly for analysis by SDS-polyacrylamide gel electrophoresis. In the labeled conditioned medium, a heterogeneous band is detected which represents the secreted colony-stimulating factor. It represents a major percent of the labeled secreted protein (approximately 1 pg/ml by antigen level). It migrates as a heterogeneous band due to heterogeneity in glycosylation. After treatment with N-glycanase, the band collapses to a tighter band which is more easily detected. ,2, In general, highest levels of expression in COS cells can be obtained with nontoxic intracellular proteins (greater than 1/zg/10 6 cells). Secreted proteins generally yield 0.3- 1 pg/ml in the conditioned medium. Lower expression levels are usually obtained with membrane-associated proteins, possibly due to the lack of membrane surface area for which they can be deposited. If the expression of the heterologus gene cannot be detected compared to the positive control, one must ensure that the vector was properly assembled. Then it is necessary to ensure that the mRNA was properly expressed. This is conveniently done by preparing RNA and analyzing it by Northern blot hybridization. When using the pMT2 vector, it is possible to use a DHFR probe and compare the level of DHFR mRNA obtained from pMT2-transfected cells to that from cells transfected with the same vector containing the insert, since they should both have a 3'-DHFR sequence provided from the pMT2 vector. If the mRNA is of the expected size and of the appropriate amount, then one must consider the possibility that the coding region may be interrupted so not to derive an intact translation product. If the RNA is not detected, then it is recommended that a different expression vector or system is tried, since it is always possible that some unforeseen reason may result in improper transcription or mRNA processing. However, examples of this actually occurring are very infrequent. Acknowledgments I gratefullythank AndrewDorner for criticallyreadingthis manuscript, MichelleWright for assistancein its preparation,and David Israel, MoniqueDavies,and PatriciaMurtha for providingthe data illustrated.