DEVELOPMENT OF A NOVEL QUANTITATIVE ASSAY FOR THE MEASUREMENT OF CHLORAMPHENICOL ACETYL TRANSFERASE (CAT) MESSENGER-RNA

Most host cells,transfected with chloramphenicol acetyl transferase (C AT) expressing plasmids display relatively low levels of constitutive CAT activity. While this is ideal to study factors that enhance gene t ranscription, decreases in CAT levels are difficult to quantitate, usi ng conventional CAT assays. Thus, investigators have used cell activat ing agents or co-transfection of the cell lines with a second enhancer plasmid to yield higher levels of CAT activity. However, such measure s can interfere with the cellular pathways studied and eventually alte r the results. To avoid this problem, our laboratory has designed an R T-PCR assay to quantitate CAT mRNA. The ability of this assay to detec t CAT mRNA but not CAT DNA demonstrates its specificity and is achieve d using a tailed oligoprimer for the reverse transcription step. This assay is able to measure the equivalent of as few as eight copies of C AT mRNA, is reproducible and relatively easy to perform. The quantitat ive capability of the assay relies on a constant production of CAT mRN A, which is achieved using permanently transfected and cloned cell lin es bearing a defined number of CAT DNA copies per cell. This assay pro vides a tool for monitoring events at the transcriptional level and th ereby complements the currently used CAT ELISA and thin-layer chromato graphy assays.