Regulate Gene Expression Levels from pLIVE® Constructs Using Alternative Cloning Strategies
Due to the presence of a second intron downstream of the multiple cloning site (MCS) in the pLIVE® Vector (Figure 1), the position of the gene of interest (GOI) stop codon can influence the level of GOI expression due to nonsense mediated decay (NMD). It is therefore possible to express different levels of the GOI using the same expression vector backbone. If the GOI stop codon is more than 50 bp upstream of the 5’ end of intron 2, NMD could be induced in the cell, resulting in decreased GOI mRNA levels in the cell. For example, a 2-3 fold decrease in luciferase expression is observed when the luciferase stop codon is more than 50 bp upstream of the start of intron 2. To avoid NMD and maximize gene expression, engineer the GOI 3’ end restriction site immediately downstream of the stop codon, and clone the fragment into the Sac I, Sac II, or Xho I sites (green arrows) of the pLIVE® Vector MCS. To take advantage of NMD and reduce expression, clone the GOI fragment containing a stop codon into the Nhe I, Asc I, Sal I, Sma I or BamH I sites (red arrows).