AAV with Enh358MCK promoter driven roGFP1

This AAV expresses roGFP1 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.

roGFP1 is a reduction-oxidation sensitive green fluorescent protein used as a biosensor. Two cysteines were introduced into the beta-barrel structure of GFP; the oxidation state, reduced dithiol or the oxidized disulfide, of the engineered thiols determines the fluorescent properties of the sensor. roGFPs (roGFP1 and roGFP2), have two fluorescent excitation maxima at about 400 and 490 nm, respectively and display rapid and reversible ratiometric changes in fluorescence in response to changes in redox potential in vitro and in vivo.