AAV with tMCK promoter driven RFP-T2A-FLPo

This AAV expresses RFP-T2A-FLPo driven by a muscle tMCK promoter.

tMCK is constructed by ligating a triple tandem copies of mouse MCK enhancer ( about200 bps each) to the ~100bp mouse MCK basal promoter.). This promoter shows extremely sensitive tissue-specificity: (1) In differentiated C2C12 myotubes, the tMCK promoter is 30-50 fold stronger than the Enh358MCK promoter and 10-20 fold stronger than the CMV promoter; (2) In muscle tissue, tMCK promoter is 15-20 fold stronger than the Enh358MCK promoter, and 3-5 fold stronger than the CMV promoter; (3) Strong tissue-specificity: in liver tissue, the expression level from tMCK promoter is only about 0.3-0.5% of that of the CMV promoter.

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).

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.