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Overview of Vector Design for Mammalian Gene Expression

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Mammalian Gene Expression Vector Design REVIEW 151 Overview of Vector Design for Mammalian Gene Expression Randal J. Kaufman* Abstract The expression of cloned genes in mammalian cells is a basic tool
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Mammalian Gene Expression Vector Design REVIEW 151 Overview of Vector Design for Mammalian Gene Expression Randal J. Kaufman* Abstract The expression of cloned genes in mammalian cells is a basic tool for understanding gene expression, protein structure, and function, and biological regulatory mechanisms. The level of protein expression from heterologous genes introduced into mammlaian cells depends upon multiple factors including DNA copy number, efficiency of transportation, mrna processing, mrna transport, mrna stability, and translational efficiency, and protein processing, transport, and stability. Different genes exhibit different rate limiting steps for efficient expression. Multiple strategies are available to obtain high level expression in mammalian cells. This article reviews vector design for expression of foreign genes in mammalian cells. Index Entries: DNA transfection; eukaryotic expression vectors; gene induction; DNA transformation. 1. Introduction The technology of expressing foreign genes in mammalian cells has become increasingly important to study a number of biological questions and as a primary method for production of proteins for pharmaceutical use. Mammalian cells are frequently used as a host for expression of foreign genes because: 1. DNA cloned from higher eukaryotic cells (both cdnas and genomic clones) is readily expressed since the signals for transcription, mrna processing, and translation are conserved in higher eukaryotic systems; 2. Proteins are expressed in a stable functional form since the machinery to facilitate proper protein folding and assembly are conserved in higher eukaryotic cells; 3. Many post-translational modifications, especially for those proteins that transit the secretory pathway, are efficiently performed; and 4. Many proteins are readily secreted from mammalian cells providing the ability to isolate the protein from conditioned medium that contains low amounts of protein when cells are propagated under serum-free conditions. Mammalian cells are used as a host for gene expression in order to: 1. Confirm that cloned genes can direct the synthesis of desired proteins; 2. Study protein structure-function relationships; 3. Isolate genes by direct screening or selecting transfected cells that express a desired protein; 4. Produce proteins that are available in limited quantity; and 5. Evaluate the physiologic consequences of expression of specific proteins in mammalian cells in order to study biological regulatory control mechanisms. The choice of a particular expression strategy is dependent upon the objectives of the study. The *Author to whom all correspondence and reprint requests should be addressed. Department of Biological Chemistry, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, MI. Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved /2000/16:2/ /$12.50 MOLECULAR BIOTECHNOLOGY 151 Volume 16, 2000 152 Kaufman Table 1 The Main Macromolecular Components of E. coli and HeLa Cells Amount per Amount per Component HeLa cell E. coli cell Total DNA 15 pg a pgb Total RNA 30 pg 0.10 pg Total protein 300 pg ( pg ( mol, of average mol, of average mw 40,000) mw 40,000) Cytoplasmic ribosomes Cytoplasmic trna molecules Cytoplasmic mrna molecules c Nuclear precursor rrna molecules Heterogeneous nuclear RNA molecules d Total dry weight 400 pg 0.4 pg a HeLa cells are hypotetraploid, i.e., they contain about four copies of each chromosome. The normal diploid human DNA complement is about 5 pg/cell. b A rapidly growing E. coli cell contains, on the average, four DNA genomes. Each genomic DNA weighs pg. c An average chain length of 1500 nucleotides is assumed. d An average chian length of 6000 nucleotides is assumed; this group of molecules contains precursor mrnas. criteria in evaluating which expression system to employ include: 1. The method desired to introduce the foreign gene into the cell; 2. The particular requirement for a specific cell type in which to obtain expression; 3. The amount of protein expression required to achieve the goals of the study; and 4. The particular need for an inducible vector to obtain expression of proteins that are potentially toxic. Two general methods for transfer of genetic material into mammalian cells are those mediated by virus infection and those mediated by direct DNA transfer. This chapter will discuss the advantages, utilities, and disadvantages of several vector systems that have proven most successful to obtain high level expression of heterologous genes in cultured mammalian cells. Although there are significant advantages for the use of mammalian cells to express foreign genes, the size of the typical mammalian cell does limit the percentage of total cell protein that can be produced through mammalian cell expression systems. Table 1 shows that the amount of DNA, RNA, and protein per cell is roughly times greater for mammalian cells than for Escherichia coli. Thus, introduction of a single gene into mammalian cells would produce approx times less of that gene product as the total cellular protein compared to a single gene in E. coli. A high level of expression in mammalian cells (approx 10 µg protein/10 6 cells/d) represents only 2.5% of the total cellular protein. 2. Expression of Exogenous Genes Transfected into Mammalian Cells 2.1. Expression by Transient DNA Transfection When cells take up DNA, they express it transiently over a period of several days to several weeks and eventually the DNA is lost from the population. The ability to express this DNA over a short period of time is called transient expression. Transient expression is a convenient and rapid method to study expression of foreign genes in mammalian cells (Table 1). The efficiency of expression after transient transfection of plasmid DNA is dependent upon the number of cells that incorporate DNA, the gene copy number, and the Mammalian Gene Expression Vector Design 153 expression level per gene. For several established cell lines it is possible to directly introduce plasmid DNA into 5 50% of the cells in the population. A variety of methods for introducing DNA have been reviewed (1). The most convenient and reproducible methods are DNA transfection mediated by DEAE Dextran, electroporation (2), and cationic phospholipids. Different labs find that one or another of these methods is generally more successful. Certain cell types may be more amenable to transfection by one procedure vs another. If one method does not work effectively it is recommended to try an alternate procedure. Transient expression offers a convenient means to compare different vectors and ensure that an expression plasmid is functional before using the expression plasmid to establish a stable expressing cell line. Transient expression experiments obviate the effects of integration sites on expression and the possibility of selecting cells that harbor mutations in the transfected DNA. Expression vectors should be tested by transient transfection prior to the more laborious procedure of isolating and characterizing stably transfected cell lines. A large variety of expression vectors for transient expression are described in the literature. Most useful vectors contain multiple elements that include: 1. An SV40 origin of replication for amplification to high copy number in COS-1 monkey cells; 2. An efficient promoter element for transcription initiation; 3. mrna processing signals that include intervening sequences and mrna cleavage and polyadenylation 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. If a relatively efficient expression vector is used, then the expression level obtained from a particular insert is most dependent upon the particular gene insert and to a lesser extent upon the particular vector. Thus, it is not possible to generalize results obtained from one insert to another. The most widely used and successful host-vector system for transient expression is based on the small DNA tumor virus, simian virus 40 (SV40). African green monkey kidney cells transformed with an origin-defective mutant of SV40 cells express high levels of the SV40 large tumor (T) antigen that 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. Generally, with the high degree of replication mediated by SV40 T-antigen, the strength of the promoter is not a major factor in limiting expression of foreign genes. Most vectors for mammalian cells contain constitutive promoter elements such as the SV40 early promoter, the Rous sarcoma virus promoter, the adenovirus major late promoter, or the human cytomegalovirus immediate early promoter that are very active in a wide variety of cell types from many species. ped is an efficient vector for transient expression in mammalian cells and can also be used to readily derive stably transfected cell lines (Fig. 1) (3). ped can be obtained from Monique Davies at Genetics Institute (Cambridge, MA). It contains plasmid sequences from puc18 which allow for propagation and selection for ampicillin resistance in E. coli. It contains the SV40 origin of replication and enhancer element and 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 for insertion of foreign DNA. In the 3' end of the transcript there is a cleavage and polyadenylation signal from the SV40 early region. This vector contains a DHFR coding region in the 3' end of the transcript. ped also contains the adenovirus virus-associated (VAI) gene, an RNA polymerase III transcription unit encoding a small RNA that inhibits the double-stranded RNA activated protein kinase (PKR). PKR is activated upon DNA transfection and it phosphorylates the alpha subunit of the eukaryotic initiation factor 2 to inhibit translation initiation (4). The VAI gene improves translation 154 Kaufman Fig. 1. Efficient cloning, selection, and expression dicistronic vectors for mammalian cells (3). The vectors containing the selection markers for wild-type DHFR (ped) and neomycin phosphotransferase (pz) are shown. of plasmid-derived mrna by inhibiting PKR activation. ped encodes a dicistronic mrna containing a dihydrofolate reductase (DHFR) coding region within the 3' end of the mrna. Efficient translation of DHFR is mediated by the internal ribosomal entry site from encephalomyocarditis virus. Thus, expression of the dicistronic mrna from this vector can be used to directly select for DHFR expression in Chinese hamster ovary cells that are deficient in DHFR (5) (see Subheading 2.3.). In many cases it is desireable to identify the transiently transfected cell within the total cell population. Several approaches have proven to be quite successful. It is possible to utilize a fluorescent derivative of methotrexate (MTX-F, obtained from Molecular Probes, Portland, OR) and stain DHFR in living cells that are transfected using the ped vector for analysis by fluorescence microscopy or by fluorescence-activated cell sorting. Fluorescence-activated cell sorting provides the ability to isolate the transfected subpopulation for direct study (6). Alternatively, the expression vector petf can be used. ETF produces a dicistronic mrna encoding the membrane surface protein tissue factor that can be stained with specific monoclonal antibody (7). In this manner, it is possible not only to identify the transfected cells, but it is also possible to isolate large numbers of transfected cells by panning with the specific antibody. It is possible to use cotransfection with an expression vector encoding the green fluorescence protein (GFP) derived from the bioluminescent jellyfish Aequorea victoria (psyn- GFPS65T can be obtained from Brian Seed, Mass. Gen Hospital [Boston, MA]) (8). This protein fluoresceses green after excitation by 395-nm light. Frequently, it is desireable to cotransfect several different plasmid DNA molecules. By titration of the amount of different plasmid DNA molecules, we have shown that DEAE-mediated dextran transfection of COS-1 cells yields approx different individual plasmid DNA molecules expressed in a single cell. Thus, cotransfection of several different plasmid DNA molecules will yield a subpopulation of cells that coexpress each plasmid DNA Inducible Expression of Foreign Genes Systems that permit stringent induction of gene expression offer unique advantages to study a diversity of biological questions as well as an approach to express gene products that are potentially cytotoxic. Sequences required for induced transcription from a number of promoters have been identified and incorporated into expression vectors that respond to a variety of stimuli such as heat shock (9), steroid hormones (10,11), heavy metal ions (12), interferon (13), iron (14), and so forth. 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 consider the fold induction and the maximal achievable Mammalian Gene Expression Vector Design 155 expression level. In many cases, the fold induction may be large but the maximal level of expression is low compared to a constitutive promoter. Attaining an efficient inducible system utilizing eukaryotic transcriptional signals has proven problematic because of the lack of tightness of control, or to pleiotropic effects caused by the inducing stimulus (for example, heat shock, heavy metal ion, stroid hormone, and so forth). Systems that are based on well-defined regulatory elements from evolutionarily distant species have proven especially useful. At present most success is obtained through the use of inducible promoters based on bacterial repressor-operator sequences that utilize either the E. coli lactose (Lac) (15) or the Tn10-derived tetracycline resistance (Tet) operon responsive repressor elements (16). Fusion proteins were constructed that were composed of the transcriptional activation domains of strong activators (such as the herpes simplex viral protein 16 immediate early gene [VP16]) and the Lac or Tet repressor proteins, respectively. In these activation systems, the effector IPTG or tetracycline prevents transcription due to inactivation of the transactivator required for transcription of a basal promoter containing Lac or Tet operators surrounding the transcription start site and because removal of the effector activates transcription (17). Recently, the Tet repressor system has been modified by fusing the VP16 activation domain with a mutant Tet repressor from E. coli. As opposed to wild-type transactivators, this mutant transactivator requires certain tetracyclines, such as doxycycline, for specific DNA binding. Thus, it is possible to directly activate transcription of the Tet operator and permit rapid induction by more than a 1000-fold (18). Most optimal use of this system requires first generating a stably transfected cell line that expresses the specific trans-activator and then transfecting into that cell the Tet operator containing the desired inducible gene Isolation of Stably Transfected Mammalian Cell Lines Selection for stable integration of plasmid DNA into the host chromosome permits the generation of stably transfected cell lines that indefinitely express a desired gene product. High-level expression of transfected DNA can be obtained through amplification of the transfected DNA by selection for a cotransfected marker gene product. Although a number of selectable amplifiable marker genes have proven useful in DNA transfer experiments, most success and experience has been using selection for dihydrofolate reductase (DHFR) genes by growth in sequentially increasing concentrations of methotrexate (Table 2). DHFR catalyzes the conversion of folate to tetrahydrofolate (FH4). FH4 is required for the biosynthesis of glycine from serine, for the biosynthesis of thymidine monophosphate from deoxyuridine monophosphate, and for purine biosynthesis. Methotrexate (MTX) is a folic acid analog that binds and inhibits DHFR, leading to cell death. When cells are selected for growth in sequentially increasing concentrations of MTX, the surviving population contains increased levels of DHFR that result from amplification of the DHFR gene. Most frequently, the degree of gene amplification is directly proportional to the expression level of DHFR. Highly resistant cells may contain several thousand fold elevated levels of DHFR. The wide utility of the DHFR selection system relies on the availability of Chinese hamster ovary (CHO) cells that are deficient in DHFR (19). Most commonly used clones are the DUKX- B11 (DXB-11) isolated from the proline auxotroph CHO-K1 and DG44 isolated from the CHO-Toronto cell lines. These lines can be obtained from Dr. Lawrence Chasin, Columbia University (New York, NY). Coamplification of heterologous genes with DHFR in DHFR-deficient CHO cells can yield cell lines that express high levels of a protein from heterologous genes. The advantages of CHO cells for the expression of heterologous genes include: 1. The amplified genes are integrated into the host chromosome and are stably maintained even in the absence of continued drug selection; 2. A variety of proteins can be properly expressed at high levels CHO cells; 3. CHO cells adapt well to growth in the absence of serum and can grow either attached or in suspension; and 156 Kaufman Table 2 Expression Levels and Utilities for Different Mammalian Cell Expression Systems Optimal Cell line Mode of DNA transfer expression level Primay utility COS-1 Transient transfection 1 µg/ml Cloning by expression Rapid characterization of clones CHO-DHFR- Stable DHFR+ transfec µg/ml High level constitutive tion and amplification expression Primate Vaccinia virus 1 10 µg/ml Expression of multiple polypeptides Vaccines 4. CHO cells can be scaled to greater than 5000 liters. A variety of selection and coamplification vectors have been constructed in which the product gene and the selection gene are contained within the same transcription unit. These dicistronic mrna expression vectors are based on the use of a picornaviral internal ribosomal entry site. Members of the picornavirus family, including poliovirus and EMC virus, have a long 5' untranslated region that mediates internal ribosome binding and cap-independent translation of mrna (20). The sequence required to promote internal initiation from encephalomyocarditis (EMC) virus extends from nucleotide 260 to 834 of the viral genome. Mammalian cell expression vectors were generated that utilize the 5' untranslated region from EMC virus to promote efficient internal translation initiation of selectable markers encoding DHFR (ped, Fig. 1), a methotrexate resistant DHFR (pemc-mtx r ), neomycin phosphotransferase (pz, Fig. 1), and adenosine deaminase (pea) (3). The use of ped is limited to DHFR-deficient cells. pz, pea, pemc-mtx r may be used as dominant markers in different cell types. These vectors permit the rapid derivation of stable cell lines that express high levels of the desired product. Since there is no selective advantage for deletion of the open reading frame contained in the 5' position, these vectors do not undergo deletion upon selection for further increases in expression. If deletion of the 5' open reading frame occurs, it is a good indication that the gene is detrimental to cell growth. 3. Vaccinia Virus Mediated Expression of Heterologous Genes in Mammalian Ce
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