Abstract
BackgroundPrevious work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line) and COS-7 (a low expressing cell line).ResultsThe main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence) are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes.ConclusionAfter exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.
Highlights
Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system [1], as compared with a 2–3 fold increase seen with previous similar systems [2]
After investigating numerous factors, including the Internal Ribosome Entry Sequence (IRES) element, T7 RNAP activity, cytosolic nuclease levels and intra-cellular processing, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in gene expression
When the system was tested in the COS-7 cell line, the autogene system resulted in levels of gene expression that were lower than the nuclear control (Figure 1)
Summary
Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system [1], as compared with a 2–3 fold increase seen with previous similar systems [2]. IRES elements were first isolated from viral genomes, such as the encephalomyocarditis virus (EMCV), where they allow the translation of viral mRNA transcripts, made in the cytoplasm,
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