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Tips from the Bench: In Vivo Tip

Express Genes In Vivo with pLIVE® Vectors

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).

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Layout of the pLIVE Vector Multiple Cloning Sites (MCS)

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 GOI stop codon is adjacent to the fragment's 3' end restriction enzyme recognition sequence.

Test the Expression of pLIVE® Construct In Vitro

Before beginning in vivo gene expression studies using a pLIVE® expression construct, verify that your expression construct is correctly expressing your GOI. Transfect your pLIVE® expression construct containing your GOI into a human liver cell line such as Hep G2. Harvest the cells 24-48 hrs post-transfection, and assay for expression of your GOI using standard western blotting procedures, or applicable assay. For example, Hep G2 cells transfected with the pLIVE®-LacZ reporter vector show robust ß-galactosidase expression (Figure 2).

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ß-galactosidase Is Expressed from pLIVE-lacZ Vector in Hep G2 Cells

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.

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