AAV with Enh358MCK promoter driven RFP-T2A-iCre

This AAV expresses RFP-T2A-iCre 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.

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).

Cre is a recombinase from the P1 bacteriophage. This enzyme carries out site-specific recombination between two DNA recognition sites (LoxP sites) through a topoisomerase I-like mechanism. The Cre recombinase here has been codon-optimized leading to a higher level of Cre protein expression (iCre). The 34base pair (bp) loxP recognition site, ATAACTTCGTATA ATGTATGC TATACGAAGTTAT, consists of two 13 bp palindromic sequence which flank an 8 bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. For example, two separate DNA species both containing loxP sites can undergo fusion as the result of Cre-mediated recombination. DNA sequences found between two loxP sites on the same DNA molecule are said to be floxed. In this case, the products of Cre-mediated recombination depend upon the orientation of the loxP sites: DNA found between two loxP sites oriented in the same direction will be excised as a circular loop of DNA, whereas DNA between two loxP sites that are oriented oppositely will be inverted. Besides LoxP sites, other Lox sites have been designed and tested, such as Lox272 and LoxN etc. These Lox elements have been widely used for genetic studies