AAV with Enh358MCK promoter driven FLPo

This AAV expresses FLPo driven by a muscle Enh358MCK promoter.

Muscle creatine kinase (MCK) is highly active in all striated muscles. MCK is the most abundant non-mitochondrial mRNA that is expressed in all skeletal muscle fiber types and is also highly active in cardiac muscle. The main regulatory regions in the murine MCK gene include a 358 bp proximal promoter and a muscle-specific, 200bp enhancer located approximately 1.1 kb away from the transcription start site. The muscle-specific enhancer contains 2 copies of E-Boxes, which are believed to be critical for the enhancer activity. Enh358MCK (Enh stands for enhancer, and 358MCK stands for the 358bp MCK proximal promoter) is constructed by ligating the ~200bp enhancer with the 358 bp proximal promoter of the murine MCK gene. This artificial promoter of about 0.6 Kb displays more or less the same activity as that of the full length 1.3 Kb MCKpromoter.

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.