Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells

Abstract

A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA

“…As control groups, apoEdeficient mice were injected with pCMV5 empty plasmid or pRC112-HCV plasmid, coding for an unrelated foreign protein. 22,28 These control groups were used in order to exclude the possibility of a non-specific mechanism by which an immune response to the vector components or the foreign transgene results in a non-specific reduction in serum cholesterol. We showed that the appearance of apoE in the blood of apoE-deficient mice injected with pCMV-E3 was associated with a dramatic reduction of serum cholesterol levels, as compared with control mice.…”

“…11 As negative controls, the pCMV5 and pRC112-HCV plasmid vectors were used. 22,28 Plasmids were grown in Escherichia coli DH5 ␣ F1Ј and prepared as described elsewhere. 22 Animals and intramuscular injections C57BL/6J apoE-deficient mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were maintained on a normal chow diet.…”

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues

“…1,2 Cytokine gene therapy approaches have been actively evaluated for a variety of potential applications in the fields of cancer immunotherapy, cancer vaccines and gene-based vaccinations against infectious diseases. [3][4][5][6][7][8][9] For many cytokine gene therapy approaches, a sharp transient gene expression peak, rather than a high and prolonged transgenic cytokine expression is valued by investigators. [3][4][5][6][7][8][9][10][11] It has also been suggested that although using current transgenic experimental systems results in low specific transgenic cytokine levels in regional or localized tissue, a 10-20 fold increase in expression levels might effectively facilitate certain physiological activities and an efficacious response, as indicated in some tumor vaccine studies.…”

“…[3][4][5][6][7][8][9] For many cytokine gene therapy approaches, a sharp transient gene expression peak, rather than a high and prolonged transgenic cytokine expression is valued by investigators. [3][4][5][6][7][8][9][10][11] It has also been suggested that although using current transgenic experimental systems results in low specific transgenic cytokine levels in regional or localized tissue, a 10-20 fold increase in expression levels might effectively facilitate certain physiological activities and an efficacious response, as indicated in some tumor vaccine studies. 12,13 Taking into consideration the aforementioned advantages of increasing transgene expression and the necessary caution of not 'overdosing' gene transfer vectors due to safety concerns, we believe that it is very important to start developing specific transgene expression systems that can be augmented by molecular biology strategies for upgrading gene expression levels.…”

“…In this experiment, cytokine genes were constructed to contain a CMV immediate/early promoter sequence 4 with either a CMV-derived intron A sequence or a short, SV40-derived splice donor/splice acceptor (SD/SA) sequence. Alternatively, cytokine transgenes were constructed under the control of a human EF-1a promoter linked with an SV40 intron-1 enhancer.…”