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Regulate pLIVE® Expression Level

pLIVE® (Liver IVivo 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.

A map showing the multiple cloning site (MCS) region of the pLIVE® plasmid and insertion sites with likelihood of inducing nonsense mediated decay (NMD).

Layout of the pLIVE® Vector Multiple Cloning Sites (MCS). The layout of the pLIVE® Vector MCS is illustrated relative to the pLIVE® Vector promoter and introns 1 and 2. Green arrows indicate cloning sites that will place the gene of interest (GOI) stop codon <50 bp* from the 5′ end of intron 2, and the red arrows indicated cloning sites that will place the GOI stop codon >50 bp from the 5′ end of the intron 2. Cloning the GOI into these sites will induce nonsense mediated decay and reduce GOI expression.

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.

Brightfield and beta-galactosidase stained Hep G2 cells after transfection with the pLIVE®-LacZ Reporter Vector using TransIT®-LT1 Transfection Reagent.

ß-galactosidase Is Expressed from pLIVE®-lacZ Vector in Hep G2 Cells. HepG2 cells were mock transfected (A) or transfected with the pLIVE®-lacZ Reporter Vector (B) using the TransIT®-LT1 Transfection Reagent. Forty-eight hours post-transfection the cells were stained with Beta-Galactosidase Staining Kit (Mirus Bio LLC) to identify the pLIVE®-lacZ transfected ß-galactosidase expressing cells.

References

  1. S. Lykke-Andersen and T. H. Jensen, Nat Rev Mol Cell Biol (2015).
    DOI: 10.1038/nrm4063

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