AAV with Enh358MCK promoter driven SEAP

This AAV expresses SEAP 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 SEAP reporter gene encodes a truncated form of the human placental alkaline phosphatase that lacks the membrane anchoring domain. Therefore, the protein can be efficiently secreted from transfected cells allowing for detection of reporter gene activity without cell lysis. Using a secreted reporter protein has several advantages over traditional reporter assays: 1) Cell lysis is not required for analysis so a single set of cells can be used for both the SEAP assay and another purpose; 2) Gene expression kinetics can be studied by the repeated collection of the culture medium from the same cultures; and 3) By changing the culture medium prior to an experiment, the assay background is reduced to an extremely low level.