Improved expression vectors for eukaryotic promoter/enhancer studies.

We describe two transfectable vectors designed to facilitate the functional analysis of eukaryotic promoter/enhancer sequences. The first, pJFCAT1, is an improved chloramphenicol acetyltransferase (CAT) reporter gene expression vector with two features that distinguish it from the majority of other CAT vectors currently in use: 1) it carries a trimer cassette of the simian virus 40 major late polyadenylation site to block plasmid-initiated read-through expression of CAT, and 2) it includes the phage f1 origin of replication, permitting generation of single-stranded copies to serve as templates for oligonucleotide-directed mutagenesis or single-strand DNA sequencing. The promoterless pJFCAT1 directs little if any CAT activity in transfected mouse L cells and, therefore, may be particularly useful for the analysis of weak promoters whose activity is otherwise masked by background CAT expression. The second vector, pTAG-1, uses human beta-globin as a reporter gene and was designed to facilitate the analysis of reporter gene expression at the RNA level. Like pJFCAT1, pTAG-1 also includes the simian virus 40 polyadenylation site trimer cassette located just upstream of the promoter insertion site. We have used each of these vectors to study functional elements in the human and mouse thymidine kinase promoters.     

Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element

A plasmid containing the adenovirus E2 gene, a gene normally requiring E1A-mediated induction during viral infection, is expressed very poorly upon transfection into mouse L cells. If the same plasmid is transfected into 293 cells, which constitutively express the adenovirus E1A gene, or into L cells together with a plasmid containing the E1A gene, the E2 gene is expressed at higher levels. Cotransfection of the E2 plasmid with a plasmid containing the pseudorabies virus (a herpesvirus) immediate early gene results in an even higher increase in the level of E2 expression. In addition, efficient E2 expression in the absence of trans induction was obtained by inserting E1A upstream promoter sequences at the 5' or 3' end of the E2 gene, indicating that these E1A sequences possess enhancer properties. Thus the efficient expression of the E2 gene can be obtained either by a structural change in the gene itself or by a trans-acting induction.     

The IE2 promoter/enhancer region from mouse CMV provides high levels of therapeutic protein expression in mammalian cells

Protein expression in mammalian cells is key for the production and manufacturing of bio-therapeutics with human-like properties and activities. As a molecular basis for reaching high protein expression levels, efficient promoter/enhancer systems are a prerequisite. Here we identify a novel enhancer from the mouse cytomegalovirus (CMV) immediate early 2 (IE2) region as a strong expression-promoting element. We further demonstrate its activity in bi-directional promoter architecture and apply it to generate production clones for IL-18BP, a protein with therapeutic indications in autoimmune diseases. These data show that the IE region from mouse CMV, and the IE2 enhancer/promoter in particular, have a broad potential for application in novel gene expression systems for research, development, and manufacturing of protein drugs.     

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.     

Optimizing gene expression in BPV-transformed cells: effects of cell type on enhancer/promoter interaction.

We have compared several combinations of enhancers and promoters in expressing the chloramphenicol acetyl transferase gene in transient assays, in mouse C127, the most widely used host cell for the bovine papilloma virus (BPV) expression vector. Of the various combinations tested, the unit comprised of the SV40 enhancer and adenovirus type 2 major late promoter (MLP) was the most active in BPV transformed C127 cells. We further demonstrate that untransformed and BPV transformed C127 cells respond differently to the various enhancer/promoter combinations tested. Moving the SV40 enhancer closer to the cap site of a complete MLP (from -414 to -107) reduced potentiation to less than half in BPV transformed cells. The level of potentiation with enhancer at either site was similar in human HeLa cells. In BPV transformed C127 cells, the SV40 enhancer and the MLP (at the -414 site) supports 4-5 fold greater levels of expression than the murine sarcoma virus (MSV) enhancer/mouse metallothionein (MT) promoter which has previously been extremely effective in BPV vectors. Our findings provide a basis for the improvement of the BPV vector system in supporting increased levels of expression of proteins of important therapeutic application.     

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

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.     

Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells.

A recombinant baculovirus containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of the Rous sarcoma virus long terminal repeat promoter and the E. coli beta-galactosidase gene under the control of the very late baculoviral polyhedrin promoter was used to determine if Autographa californica nuclear polyhedrosis virus, a baculovirus of Lepidoptera, can enter and express viral DNA in dipteran (Drosophila sp.) and mammalian (Mus sp.) cells that are considered refractory to baculovirus replication. Following infection, CAT gene expression was observed in both dipteran and mammalian cells, but expression in the mammalian cell line was less than 0.05% of that observed in either dipteran or lepidopteran cells. Although the level of CAT gene expression was similar in permissive lepidopteran and nonpermissive dipteran cells, expression of beta-galactosidase activity from the late polyhedrin promoter in dipteran or mammalian cells was less than 0.3% of the levels observed in lepidopteran cells. These results indicate that foreign gene expression in nonpermissive cells is promoter dependent and that late viral gene expression is restricted in these cells. The Rous sarcoma virus long terminal repeat allows substantial CAT gene expression in both a D. melanogaster cell line and Aedes aegypti midgut cells. Baculovirus DNA undergoes a limited number of replications in Drosophila cells. The results are relevant to baculovirus host range, the safety of baculoviruses as pesticides, and the development of baculovirus pesticides with expanded host ranges.