Human immunodeficiency virus vectors for inducible expression of foreign genes.

Tat-dependent expression of an endogenous lethal or deleterious foreign gene might be useful for abrogating the production of human immunodeficiency virus (HIV) from cells. This type of HIV-induced cellular killing, as well as other approaches to gene therapy for HIV infection, would be facilitated by simple HIV vectors that express introduced genes in a Tat-inducible manner. As part of studies to examine the feasibility of this concept, we constructed HIV-1 vectors that express the hygromycin B phosphotransferase gene (Hygr) in a Tat-dependent manner. Comparison of the efficiency of propagation of each vector indicates that sequences extending into the gag open reading frame are necessary in cis for efficient vector propagation. Southern blot analysis of genomic DNA isolated from vector-infected cells demonstrated that the vectors were capable of being propagated as expected without gross rearrangements or deletions. A fragment of the influenza A virus hemagglutinin (H5 HA) gene, capable of eliciting antibody and cytotoxic T-cell responses, was used as a marker for further characterization of the vector system. A Tat-dependent vector conferring the H5 HA+ phenotype was assayed by indirect immunofluorescence, and cells which contained but did not express the H5 HA gene were isolated. The activation of H5 HA expression following HIV infection of Tat- cells that stably contained but did not express the H5 HA construct was determined to be an efficient process.     

Inducible vectors for expression in mammalian cells.

The most recent developments in mammalian cell inducible expression systems have involved the use of bacterial gene control elements and viral transactivator proteins. The combination of hybrid viral transactivator and bacterial repressor proteins, and simple chemical inducers can provide induction ratios of over 1000-fold. These developments will have applications in both cell-based research and the generation of transgenic animals.     

Transfer and expression of foreign genes in mammalian cells

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.     

Episomal vectors for gene expression in mammalian cells.

An important reason for preferring mammalian cells for heterologous gene expression is their ability to make authentic proteins containing post-translational modifications similar to those of the native protein. The development of expression systems for mammalian cells has been ongoing for several years, resulting in a wide variety of effective expression vectors. The aim of this review is to highlight episomal expression vectors. Such episomal plasmids are usually based on sequences from DNA viruses, such as BK virus, bovine papilloma virus 1 and Epstein-Barr virus. In this review we will mainly focus on the improvements made towards the usefulness of these systems for gene expression studies and gene therapy.     

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.     

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.     

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37‐nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia‐based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small‐scale expression in 10⁶ cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.     

Retroviral vectors--an effective system for transfer and expression of foreign genes in mammalian cells

The review describes the use of retroviruses as vectors for transfer and expression of cloned genes. The advantages of various retrovirus-based vector systems are discussed in terms of comparison with other conventional methods of introduction of foreign genes into cultured cells.     

Persistent expression of foreign genes in cultured hepatocytes: expression vectors

We have optimized a liposome-based transfection method that mediated highly efficient stable expression of foreign genes in hepatocytes. Moreover, we have observed that the metallothionein 1 promoter in the bovine papilloma virus-based expression vector drove the highest expression of foreign genes in hepatocytes as compared with the cytomegalovirus and the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoters in the pcDNA 3-based expression vector. The cytomegalovirus promoter failed to yield significant expression in these cells. Furthermore, expression of foreign genes persisted up to at least 15 passages when expression was under the control of either the EF-1alpha or the metallothionein 1 promoter. Thus, these two promoters led to comparable stability of foreign genes in hepatocytes, with the metallothionein 1 promoter yielding a higher level of expression of foreign genes in these cells.     

Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast

New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described. One vector, pYEULlacZ, is based on pUC19 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by lacZ inactivation. In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains. The presence of the leu2-d gene appears to promote replication at high copy numbers. The introduction of CUP1 cassettes allows these plasmids to direct Cu(2+)-regulated production of foreign proteins in yeast. We show the production of a helminth antigen as an example of the vector application.     

Recombinant baculoviruses as expression vectors for insect and mammalian cells.

Baculovirus expression vectors are widely used for expressing heterologous proteins in cultured insect cells. Recent advances include further development of the system for production of multi-subunit protein complexes, co-expression of protein-modifying enzymes to improve heterologous protein production, and additional applications of baculovirus display technology. The application of modified baculovirus vectors for gene expression in mammalian cells continues to expand.