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Publisher Summary This chapter presents chloramphenicol acetyltransferase (CAT) as a gene expression tool, the system has been widely employed to characterize transfection techniques for tissues and cell lines, for characterizing DNA mutagenesis and repair systems, for genetic and biochemical analyses of transcription factors and transcription initiation complexes, for characterization of signaling pathways, for characterizing the tissue specificity of promoters in transgenic animals, and for evaluating the efficacy of delivery systems for gene therapy in intact animals. The chapter focuses on three types of CAT assay methods, a thin-layer chromatography (TLC) assay, a mixed phase assay, and a high-performance liquid chromatography (HPLC) assay. Parameters that can influence the sensitivity and reproducibility of the assays include the concentration and stability of the reagents, the temperature of the reaction, the reaction time, the extraction efficiency of reaction products in solvents, the presence of other acetylases in the reaction, the reaction volume, the type of cell transfected, the preparation of cell extracts, and the amount of extracts used in the reaction. |
