AAV with CAG promoter driven FLPo

This AAV expresses FLPo driven by an ubiquitous CAG promoter.

The roughly 1.8Kb CAG promoter (also known as CBA promoter or CAGGS promoter) is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian cells. The CAG promoter is composed of the following regulatory elements: (C) cytomegalovirus (CMV) early enhancer element; (A) the promoter region, the first exon, and the first intron of chicken beta-Actin gene, and (G) the splice acceptor of the rabbit beta-Globin gene. Like the EF1a promoter, the CAG promoter is commonly used as an alternative for the CMV promoter, from which the expression is decreased due to methylation/silencing. In many cell types tested, the CAG and EF1a promoters give much higher levels of expression than other commonly used cellular promoters such as the UBC and PGK promoters.

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.