AAV with ALB(1.4) promoter driven EGFP-T2A-CreERT2

This AAV expresses EGFP-T2A-CreERT2 driven by a liver ALB(1.4) promoter.

The ALB(1.4) is a shorter version of the ALB(1.7) promoter with a total length of 1.4 Kb. In mouse liver cells, the transgene expression from this ALB(1.4) synthetic promoter is about 3 times lower than that of ALB(1.7) promoter but 10-100 fold greater than that of the CMV promoter 10-20 weeks after plasmid injection into mouse liver.

The short 2A peptide sequences, when cloned in-frame between two genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. This differs from conventional approaches for multiple protein expressions, such as IRES-mediated bicistronic gene expression, which has several limitations including imbalanced protein expression. The use of 2A peptide sequences alleviates these concerns, since 2A-mediated “self-cleavage” gives rise to a 1:1 ratio of the two separate proteins. Several forms of 2A peptide are commonly used: T2A (Thoseaasigna virus 2A), P2A (porcine teschovirus-1 2A), E2A (equine rhinitis A virus), and F2A (foot-and-mouth disease virus, FMDV 2A).

A mutated form of estrogen ligand-binding domain (ERT2) that binds to synthetic antagonists (such as tamoxifen or its derivative 4-hydroxy-tamoxifen) but not to circulating estrogen, was fused to the Cre recombinase (Cre) to create CreERT2 (also known as “inducible Cre”). When tamoxifen binds to CreERT2, it induces a conformational change of CreERT2, leading to its nuclear translocation, followed by Cre/Lox-mediated recombination.