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Alkaline phosphatase as a reporter enzyme.
K Yoon1, M A Thiede, G A Rodan
1Department of Bone Biology and Osteoporosis Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Gene
|June 15, 1988
Summary
Alkaline phosphatase (AP) is an effective reporter enzyme for mammalian cell transfection. Its convenient detection methods, including histochemical staining, offer sensitivity comparable to radioactive assays for monitoring gene expression.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Reporter enzymes are crucial for monitoring gene expression and transfection efficiency.
- Alkaline phosphatase (AP) is a well-characterized enzyme with potential reporter applications.
Purpose of the Study:
- To evaluate alkaline phosphatase (AP) as a reporter enzyme in mammalian cell transfection.
- To compare the sensitivity of AP detection with established reporter systems like chloramphenicol acetyltransferase (CAT).
Main Methods:
- Constructed a plasmid with rat AP cDNA under the SV40 early promoter.
- Transfected mammalian cell lines (CHO, COS, ROS 25/1) with the AP plasmid.
- Measured AP activity using spectrophotometric and histochemical assays.
- Performed co-transfection with CAT plasmids for comparative analysis.
Main Results:
- AP expression resulted in 40-400-fold increase in enzyme activity above background.
- Spectrophotometric detection of AP activity was as sensitive as radioactive CAT detection.
- Histochemical staining allowed for visualization and estimation of transfection efficiency.
Conclusions:
- Alkaline phosphatase (AP) serves as a sensitive and convenient reporter enzyme for mammalian cell transfection.
- AP's ectoenzyme nature facilitates cell visualization and transfection efficiency assessment.
- The straightforward detection methods make AP a valuable tool in molecular biology research.