QUANTIFICATION OF GENE-EXPRESSION WITH A SECRETED ALKALINE-PHOSPHATASE REPORTER SYSTEM

The cDNA encoding secreted alkaline phosphatase (SEAP) is a useful too l for investigating the function of known or putative enhancer/promote r elements. SEAP has the unusual properties of extreme heat stability and resistance to the phosphatase inhibitor L-homoarginine. Therefore, endogenous alkaline phosphatase activity in transfected cells can be minimized by pretreatment of samples at 65 degrees C and incubation wi th the inhibitor. With the use of the chemiluminescent substrate CSPD( R), 10(-13) g of enzyme can be detected in culture medium, and the enz yme activity can be detected as early as 24 h after transfection. The chemiluminescence-based SEAP assay is about 10-fold more sensitive tha n similar assays using firefly luciferase as the reporter enzyme. The SEAP activity can also be assayed with a fluorescent substrate MUP, wh ich provides sensitivity comparable to luciferase. Since the enzyme is secreted to culture medium, the enzyme assay can be performed on smal l samples of the culture supernatant. Because preparation of cell lysa tes is not required, assaying for SEAP activity is faster and more con venient than assaying for intracellular reporters. Furthermore, becaus e the transfected cells are not disturbed by the sampling procedure, t he same cultures can be repeatedly sampled for time-course studies or used for further investigations.