Alkaline phosphatase as a reporter enzyme.
This study examines the use of alkaline phosphatase (AP) as a reporter enzyme. We constructed a plasmid containing the cDNA which encodes the bone/liver/kidney rat AP under the control of the simian virus 40 (SV40) early promoter and used it to transfect Chinese hamster ovary, SV40-transformed African Green Monkey kidney 7, and rat osteosarcoma 25/1 mammalian cells. AP activity in these cells, measured three days later, was 40-400-fold above background. When AP and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected, the detection of AP activity by a simple spectrophotometric assay was at least as sensitive as the detection of CAT activity using a radioactive substrate. Moreover, since mammalian AP is a membrane-bound ectoenzyme, transfected cells can be visualized by histochemical staining. This approach was used to estimate transfection efficiency. The convenient methods for AP detection should make it a useful reporter enzyme.
Yoon
K
K
Department of Bone Biology and Osteoporosis Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Thiede
M A
MA
Rodan
G A
GA
D000123:Acetyltransferases / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase; D005822:Genetic Vectors; D006651:Histocytochemistry; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D010957:Plasmids; D013053:Spectrophotometry; D014162:Transfection
D008565:Membrane Proteins; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase