Investigation of factors responsible for cell line cytoplasmic expression differences

The 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.After 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.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]. 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.Cytosolic nucleases have been discovered and characterized [3,4]. These proteins have found to be an important factor with respect to the cytosolic half-life of plasmid based vectors. These nucleases could be important to researchers using plasmid based delivery systems, especially cytoplasmic gene expression systems.Internal Ribosome Entry Sequences (IRES) are elements that have been shown to drive expression of the second gene in a bi-cistronic mRNA transcript, as well as increase the translation of un-capped transcripts. 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, and thus lacking the 5' cap structure essential for mRNA translation [5]. Various viral IRES elements, such as the EMCV, FMDV (foot and mouth disease virus), and other picornavirus based IRES elements share sim