Plasmid pLTRpoly: a versatile high-efficiency mammalian expression vector.

Plasmid pLTRpoly is an expression vector enabling high-level expression of introduced genes in a variety of cell types. A large multiple cloning site (MCS) and the availability of the full-length nucleotide sequence facilitate the generation of constructs using this vector. Here, constructs made with pLTRpoly have been tested in NIH3T3 mouse fibroblasts.     

Overview of vector design for mammalian gene expression

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.     

A vector, pSHT, for the expression and secretion of protein domains in mammalian cells.

A phagemid (pSHT) containing the pUC and M13 ori sequences was constructed to facilitate the expression of partial cDNAs or of sequences encoding mammalian membrane- and secretory-protein domains. It provides a start codon and signal sequence flanked upstream by the simian virus 40 and bacteriophage T7 promoters and downstream by cloning sites, stop codons in all three frames, splicing and polyadenylation signals.     

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

Mutagenesis of a shuttle vector plasmid in mammalian cells.

Recently we and others have reported a high frequency of mutagenesis of shuttle vector plasmids after passage in mammalian cells (Razzaque et al., Proc. Natl. Acad. Sci. U.S.A. 80:3010-3014, 1983; Calos et al., Proc. Natl. Acad. Sci. U.S.A. 80:3015-3019, 1983). The mutation frequency was determined by monitoring the integrity of a bacterial marker gene on the plasmid by standard microbiological procedures. Mutant plasmids contained deletions, insertions of cell DNA, and point mutations. The observed mutation frequency of 1% is much higher than that of cellular markers and could be due to the induction of a mutagenic environment by infection with a replicating plasmid. Alternatively, the hypermutagenesis may be due to some critical transient or persistent difference between the DNA in the plasmid and the cellular chromosome. We performed a number of experiments designed to distinguish between these alternatives, with particular reference to deletion mutagenesis. We conclude that mutagenesis was specific to the plasmid and propose that the majority of the deletion and insertion mutants were generated very early in the infection, before replication of the vector. However, some deletion mutagenesis also occurred after plasmid replication had begun.     

Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.     

A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.     

A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.     

A mammalian expression vector for expression and purification of secreted proteins for structural studies

A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined.