One reporter for multiple profiling of TFs

One reporter for multiple profiling of TFs


Some days ago I was challenging the concept that a reporter need to be a protein. Imagine my surprise in reading the March issue of Nature Methods, where Romanov and colleagues from Attagene Inc, describe a novel method for simultaneously reporting on the activity of a large panel of transcription factors. Here the reporter is a synthethic sequence that isn't translated, but only transcribed, an example of RNA-based reporters. Moreover, the paper breaks the dogma 'one promoter - one reporter' by analyzing activities of multiple TFs with just 'one' reporter sequence.

The trick is that they cloned TFs binding sites in front of a reporter sequence with an enzymatic cleavage site in a separate position for each TF, so that after these constructs are transfected within a cell, digest of the reporter species produced spectrum of DNA fragments that mirrored TF activities profile.

To be more technical, for multiplexed detection of transcription factors the "Factorial(TM)" 30 TF reporter library is introduced into cells of interest by using standard transfection procedure. Some time after transfection, total cellular RNA is isolated and reversely transcribed. The reporter cDNAs are amplified by PCR using a pair of primers that are common for all reporters. The PCR products are labeled with a fluorescent dye and digested by the HpaI. The digestion produces a spectrum of fluorescently labeled DNA fragments of different lengths that are resolved by capillary electrophoresis (CE), detected as separate fluorescent peaks and then quantified.

Although CE is limited by a narrow dynamic range, and the assay is challenged by the management of up to 30 sequence transfection in the same cell culture, this novel technique offers a valuable alternative for TF activity fingerprinting that outclasses classic TF binding ELISAs for significance and elegance.

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Romanov, S., Medvedev, A., Gambarian, M., Poltoratskaya, N., Moeser, M., Medvedeva, L., Gambarian, M., Diatchenko, L., & Makarov, S. (2008). Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors Nature Methods, 5 (3), 253-260 DOI: 10.1038/nmeth.1186