AAV with tMCK promoter driven SEAP

This AAV expresses SEAP 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 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.