Development of a prokaryotic expression vector that exploits dicistronic gene organization.

For unknown reasons, levels of expression of foreign genes inserted into expression vectors in Escherichia coli have frequently been undetectable. The most critical step in the successful production of foreign proteins seems to be the initiation of translation. Since most prokaryotic genes are transcribed in a polycistronic form, we have devised a new prokaryotic expression system utilizing dicistronic gene organization. Downstream from a strong promoter and the gene encoding glutathione S-transferase from Schistosoma japonicum, various foreign genes were connected via a ribosome-binding site, a stop codon and a start codon. The VH domain of an immunoglobulin fused to the alpha subunit of tryptophan synthase, FK506-binding protein, cyclophilin, and a domain of a major histocompatibility complex antigen were successfully produced in E. coli as discrete polypeptides by this method.     

Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen

Flow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts the relative expression level of the therapeutic protein for each clone. This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96-well plate clone screening and elimination of unstable clones at an earlier stage in the development process. Furthermore, because this method does not rely on the availability of an antibody specific for the therapeutic protein being expressed, it can be easily implemented into any cell line development process.     

Neutral selection of transfected mammalian cells using tissue plasminogen activator gene expression

The gene that produces the proteolytic enzyme, tissue plasminogen activator (tPA), was used as a selectable marker for transfection. Cells that were successfully transfected with plasmids containing a gene for tPA (tpa) produce plaques in the caseinolysis assay. Two tpa-containing plasmids were constructed and used to transfect a variety of cell types. One plasmid contained the promoter region of simian virus 40 and the other contained the Moloney murine leukemia virus promoter. No significant difference in transfection frequencies was found for the two plasmids. However, 80% of the cell types tested produced plaques. This system allows for the identification and isolation of transfected cells under conditions that are not lethal to nontransfected cells.     

Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells.

The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.     

Cell-surface streptavidin fusion protein for rapid selection of transfected mammalian cells

We developed a series of eight mammalian cell surface marker fusion genes by using the streptavidin gene from Streptomyces avidinii. These fusion genes are useful and non-growth-toxic selection markers for rapid-harvest transfected mammalian cells. Two streptavidin constructs were used; the longer fragment contains the native bacterial signal sequence, which the shorter fragment lacks. For expression of the streptavidin antigen on the surface of mammalian cells, streptavidin was flanked by a mammalian signal sequence and a transmembrane domain (from mouse H2-K or Kit); some constructs also contained the gene for enhanced green fluorescent protein (EGFP). We transfected a series of plasmids encoding the fusion proteins into HeLa cells and determined that the transfected cells produced the fusion protein on their cell surfaces. To separate transfected cells from nontransfected cells, we incubated cells with a polyclonal antibody against streptavidin, and antibody-bound cells were harvested by the use of paramagnetic beads coupled with the corresponding secondary antibody. We obtained highly pure populations of transfected cells; this result also confirmed the production of the fusion protein on the cell surface. Cell growth assays revealed that none of the transfected fusion genes or their products adversely affected the proliferation of HeLa cells. Our results indicate that the fusion constructs we developed and the immunomagnetic separation protocol we used are valuable tools for various transfection applications. In particular, the constructs containing EGFP are advantageous because transfection efficiency can be assessed without additional treatment of cells.     

Bacteriophage T4, a new vector for the expression of cloned genes

The amino-terminal portion of the T4 rIIB gene has been fused to the coding sequence of a truncated lacZ gene from Escherichia coli, giving rise to a fusion protein with beta-galactosidase activity. The 3192-bp rIIB-lacZ gene fusion was transferred into phage T4, and enzymatically active protein was produced after phage infection. T4 may be a useful expression vector in special circumstances, in particular for proteins whose accumulation in E. coli is limited by sensitivity to proteases.     

Short-term, high-efficiency expression of transfected DNA.

We have achieved high-efficiency uptake and expression of foreign DNA in mouse Ltk- cells by modifying the DEAE-dextran-mediated transfection method of McCutchan and Pagano (J. Natl. Cancer Inst. 42:351-357, 1968) to include an initial incubation at elevated pH followed by a shock treatment with dimethyl sulfoxide. Up to 80% of mouse Ltk- cells transfected with the herpes simplex virus thymidine kinase gene expressed thymidine kinase as measured by autoradiography.     

Alteration of endogenous glutathione peroxidase, manganese superoxide dismutase, and glutathione transferase activity in cells transfected with a copper-zinc superoxide dismutase expression vector. Explanation for variations in paraquat resistance

Transfection of a human pSV2 (copper-zinc) superoxide dismutase expression vector into murine fibroblasts resulted in stable clones producing increased amounts of copper-zinc superoxide dismutase. A marked increase in endogenous glutathione peroxidase activity (up to 285%) and a smaller increase in glutathione transferase activity (up to 16%) also occurred. Manganese superoxide dismutase activity was decreased in all clones, whereas catalase and NADPH reductase activities were not affected. Alterations in glutathione peroxidase and manganese superoxide dismutase activities correlated with increases in copper-zinc superoxide dismutase activity. Whereas all clones were resistant to paraquat, a direct correlation between copper-zinc superoxide dismutase activity and resistance to paraquat did not exist. In agreement with previous reports clones expressing the highest copper-zinc superoxide dismutase activity did not display the highest resistance to paraquat. However, there was a direct correlation between the increase in glutathione peroxidase activity and paraquat resistance (p less than 0.002).     

Unstable expression and amplification of a transfected oncogene in confluent and subconfluent cells.

NIH 3T3 cells were transfected with a plasmid containing the transforming gene, v-src, from Rous sarcoma virus. One of the transformed cell lines isolated reverted to a flat, nontransformed morphology after cloning through soft agar. This cell line did not express the src gene and could no longer grow in soft agar. When these cells were held at confluence, spontaneous foci appeared which eventually covered the dish. The appearance of foci correlated with an increase in v-src gene expression, ability to grow in soft agar, and tumorigenicity in mice. When these transformed cells were trypsinized and held at subconfluence, both v-src expression and the transformed phenotype were progressively lost. Whereas rearrangement of the transfected gene was not detected, the gene copy number in the transformed cells was markedly increased (greater than 50-fold). Confluence-dependent gene amplification and deamplification have been retained after several cycles of growth alternately at high and low density, in cells recloned through soft agar, and after cells had been maintained continuously at high or low density. The results suggest that, in this cell line, reversible gene amplification plays a central role in expression of the transfected gene.     

Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries

As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.     

A GFP-based method facilitates clonal selection of transfected CHO cells

The identification of highly expressing clones is a crucial step in the development of cell lines for production of recombinant proteins. Here we present a method based on the co-expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site and thus are transcribed into the same mRNA but are translated independently. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein for each clone. By expressing recombinant growth factors in CHO cells, we demonstrate the robustness and performance of this technique. The method is an alternative to the identification of high-producer clones using various cell sorting methods, as it can be performed with standard laboratory equipment.     

A dicistronic construct allows easy detection of human CFTR expression from YAC DNA in human cells.

We have made a dicistronic construct where the picornaviral internal ribosome-entry site (IRES) driving the expression of the beta-geo gene has been inserted into the 3'untranslated region of the human CFTR gene present in a YAC. When introduced into the human cell line Caco-2 expressing the CFTR gene, the expression of the dicistronic gene can be detected by lacZ staining and follows the accumulation of the endogenous CFTR mRNA upon differentiation of the cells. These data demonstrate that this IRES-based approach presents an alternative to mRNA in situ hybridisation and allows detection of expression in an autologous system.