Overview of Vector Design for Mammalian Gene Expression Randal J

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Overview of Vector Design for Mammalian Gene Expression Randal J Mammalian Gene Expression Vector DesignREVIEW 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 transla- tional efficiency, and protein processing, transport, and stability. Different genes exhibit different rate lim- iting 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 4. Many proteins are readily secreted from mam- The technology of expressing foreign genes in malian cells providing the ability to isolate the mammalian cells has become increasingly impor- protein from conditioned medium that contains tant to study a number of biological questions and low amounts of protein when cells are propa- as a primary method for production of proteins gated under serum-free conditions. for pharmaceutical use. Mammalian cells are fre- Mammalian cells are used as a host for gene quently used as a host for expression of foreign expression in order to: genes because: 1. Confirm that cloned genes can direct the syn- 1. DNA cloned from higher eukaryotic cells thesis of desired proteins; (both cDNAs and genomic clones) is readily 2. Study protein structure-function relationships; expressed since the signals for transcription, 3. Isolate genes by direct screening or selecting mRNA processing, and translation are con- transfected cells that express a desired protein; served in higher eukaryotic systems; 4. Produce proteins that are available in limited 2. Proteins are expressed in a stable functional quantity; and form since the machinery to facilitate proper 5. Evaluate the physiologic consequences of protein folding and assembly are conserved in expression of specific proteins in mammalian higher eukaryotic cells; cells in order to study biological regulatory 3. Many post-translational modifications, espe- control mechanisms. cially for those proteins that transit the secre- The choice of a particular expression strategy tory pathway, are efficiently performed; and 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 Medi- cal Institute, University of Michigan Medical Center, Ann Arbor, MI. Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2000/16:2/151–160/$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 pga 0.017 pgb Total RNA 30 pg 0.10 pg Total protein 300 pg (5 × 109 0.2 pg (3 × 106 mol, of average mol, of average mw 40,000) mw 40,000) Cytoplasmic ribosomes 4 × 106 3 × 104 Cytoplasmic tRNA molecules 6 × 107 4 × 105 Cytoplasmic mRNA moleculesc 7 × 105 4 × 103 Nuclear precursor rRNA molecules 6 × 104 Heterogeneous nuclear RNA moleculesd 1.6 × 105 Total dry weight 400 pg 0.4 pg aHeLa cells are hypotetraploid, i.e., they contain about four copies of each chromosome. The normal diploid human DNA complement is about 5 pg/cell. bA rapidly growing E. coli cell contains, on the average, four DNA genomes. Each genomic DNA weighs 0.0044 pg. cAn average chain length of 1500 nucleotides is assumed. dAn average chian length of 6000 nucleotides is assumed; this group of molecules contains precursor mRNAs. criteria in evaluating which expression system to RNA, and protein per cell is roughly 100–1000 employ include: times greater for mammalian cells than for Esche- richia coli. 1. The method desired to introduce the foreign Thus, introduction of a single gene gene into the cell; into mammalian cells would produce approx 100– 2. The particular requirement for a specific cell 1000 times less of that gene product as the total type in which to obtain expression; cellular protein compared to a single gene in 3. The amount of protein expression required to E. coli. A high level of expression in mammalian 6 achieve the goals of the study; and cells (approx 10 µg protein/10 cells/d) represents 4. The particular need for an inducible vector to only 2.5% of the total cellular protein. obtain expression of proteins that are poten- 2. Expression of Exogenous Genes tially toxic. Transfected into Mammalian Cells Two general methods for transfer of genetic 2.1. Expression by Transient DNA material into mammalian cells are those mediated Transfection by virus infection and those mediated by direct DNA transfer. This chapter will discuss the advan- When cells take up DNA, they express it tran- tages, utilities, and disadvantages of several vec- siently over a period of several days to several tor systems that have proven most successful to weeks and eventually the DNA is lost from the obtain high level expression of heterologous population. The ability to express this DNA over genes in cultured mammalian cells. a short period of time is called “transient expres- Although there are significant advantages for sion.” Transient expression is a convenient and the use of mammalian cells to express foreign rapid method to study expression of foreign genes genes, the size of the typical mammalian cell does in mammalian cells (Table 1). The efficiency of limit the percentage of total cell protein that can expression after transient transfection of plasmid be produced through mammalian cell expression DNA is dependent upon the number of cells that systems. Table 1 shows that the amount of DNA, incorporate DNA, the gene copy number, and the MOLECULAR BIOTECHNOLOGY Volume 16, 2000 Mammalian Gene Expression Vector Design 153 expression level per gene. For several established The most widely used and successful host-vec- cell lines it is possible to directly introduce plas- tor system for transient expression is based on the mid DNA into 5–50% of the cells in the popula- small DNA tumor virus, simian virus 40 (SV40). tion. A variety of methods for introducing DNA African green monkey kidney cells transformed have been reviewed (1). The most convenient with an origin-defective mutant of SV40 cells and reproducible methods are DNA transfection express high levels of the SV40 large tumor (T) mediated by DEAE Dextran, electroporation (2), antigen that is required to initiate viral DNA rep- and cationic phospholipids. Different labs find lication. The T-antigen-mediated replication can that one or another of these methods is generally amplify the plasmid copy number to greater than more successful. Certain cell types may be more 10,000 per cell. This large copy number results in amenable to transfection by one procedure vs high expression levels from the transfected DNA. another. If one method does not work effectively Generally, with the high degree of replication it is recommended to try an alternate procedure. mediated by SV40 T-antigen, the strength of the Transient expression offers a convenient means promoter is not a major factor in limiting expres- to compare different vectors and ensure that an sion of foreign genes. Most vectors for mamma- expression plasmid is functional before using the lian cells contain constitutive promoter elements expression plasmid to establish a stable express- such as the SV40 early promoter, the Rous sar- ing cell line. Transient expression experiments coma virus promoter, the adenovirus major late obviate the effects of integration sites on expres- promoter, or the human cytomegalovirus imme- sion and the possibility of selecting cells that har- diate early promoter that are very active in a wide bor mutations in the transfected DNA. Expression variety of cell types from many species. vectors should be tested by transient transfection pED is an efficient vector for transient expres- prior to the more laborious procedure of isolating sion in mammalian cells and can also be used to and characterizing stably transfected cell lines. readily derive stably transfected cell lines (Fig. 1) A large variety of expression vectors for tran- (3). pED can be obtained from Monique Davies sient expression are described in the literature. at Genetics Institute (Cambridge, MA). It con- Most useful vectors contain multiple elements tains plasmid sequences from puc18 which allow that include: for propagation and selection for ampicillin resis- tance in E. coli. It contains the SV40 origin of rep- 1. An SV40 origin of replication for amplification lication and enhancer element and utilizes the to high copy number in COS-1 monkey cells; adenovirus major late promoter for transcription 2. An efficient promoter element for transcription initiation. Contained within the 5' end of the initiation; mRNA is the tripartite leader from adenovirus late 3. mRNA processing signals that include inter- mRNA and a small intervening sequence. There vening sequences and mRNA cleavage and are several cloning sites including for insertion of polyadenylation sequences; foreign DNA. In the 3' end of the transcript there 4. Polylinkers that contain multiple endonu- clease restriction sites for insertion of foreign is a cleavage and polyadenylation signal from the DNA; and SV40 early region. This vector contains a DHFR 5. Selectable markers that can be used to select cells coding region in the 3' end of the transcript. pED that have stably integrated the plasmid DNA. also contains the adenovirus virus-associated (VAI) gene, an RNA polymerase III transcription If a relatively efficient expression vector is unit encoding a small RNA that inhibits the used, then the expression level obtained from a double-stranded RNA activated protein kinase particular insert is most dependent upon the par- (PKR).
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