pLIVE® (Liver In Vivo Expression) is a plasmid designed for high level, prolonged expression of transgenes in the mouse liver. Thanks to a phenomenon known as nonsense-mediated decay (NMD), the expression level of genes cloned into the pLIVE® plasmid can be modulated. In short, NMD is a mechanism by which cells surveil and remove potentially deleterious mRNA during translation. In some cases, NMD is triggered when a stop codon is close to and upstream of an intron. See [1] for a review of NMD.
Due to the presence of a second intron downstream of the multiple cloning site in the pLIVE® plasmid (see figure below), the position of the gene of interest (GOI) can influence the level of its expression due to NMD. Therefore, it is possible to express different levels of the GOI using the same pLIVE® backbone.
If the stop codon of the GOI is > 50 bp upstream of the 5' end of Intron 2, NMD could be induced in the cell, resulting in decreased levels of the GOI mRNA. For example, we have observed a two- to three-fold decrease in luciferase expression when the luciferase gene’s stop codon was > 50 bp upstream of the start of Intron 2. In the figure below, the red arrows indicate 3' restriction enzyme cut sites that would position the stop codon of the GOI > 50 bp from the end of Intron 2.
Conversely, to avoid NMD and maximize gene expression, ensure the stop codon of the GOI is < 50 bp upstream of the 5' end of Intron 2. In the figure below, the green arrows indicate 3' restriction enzyme cut sites that would position the stop codon of the GOI < 50 bp from the end of Intron 2 (*provided that the stop codon is directly adjacent to the restriction enzyme recognition sequence).
To take advantage of NMD to modulate gene expression, experiment with the position of the stop codon of the GOI relative to Intron 2. Before beginning in vivo gene expression studies, verify that your pLIVE® expression construct is correctly expressing your GOI in vitro . You can do so by transfecting a human liver cell line, e.g. Hep G2, with your pLIVE® expression construct. The figure below shows an example of Hep G2 cells that have been transfected with the control pLIVE®-lacZ Vector (MIR 5520). Robust ß-galactosidase expression can be observed.