AAV with ALB(1.0) promoter driven EGFP-T2A-WGA-FLPo

This AAV expresses EGFP-T2A-WGA-FLPo driven by a liver ALB(1.0) promoter.

The ALB(1.0) is a shorter version of the ALB(1.4) promoter with a total length of 1.0 Kb. In mouse liver cells, the transgene expression from this ALB(1.0) synthetic promoter is about 3-5 times lower than that of ALB(1.4) promoter but 10-20 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).

Wheat germ agglutinin (WGA) is a transcellular tracer protein. It is commonly used in combination with another protein, such as Cre recombinase, to allow movement of the protein between cells

FLPo is a codon-optimized version of FLPe that greatly increases the protein expression and the FRT recombination efficiency in mouse cells. FLP is a site-specific recombinase (SSR) from the _ integrase family which recognizes distinct 34 bp FRT sites. Like Cre/LoxP system, the FLP/FRT system has been widely used for gene expression and generating conditional knockout mice, mediated by the FLP/FRT system. Initial use of FLP in mammalian cells revealed inefficient recombinase activity due to thermal instability of the FLP protein. Subsequent screening for thermostable mutants resulted in the identification of FLPe which has a 4-fold higher recombination efficiency at 37oC than original FLP. However, the recombination efficiency of FLPe in cells remains very low relative to other recombinases because of its non-mammalian origin. FLPo is engineered by codon optimization of FLPe, and gives much higher expression and activity than FLPe.