- High-content screening with fluorescent proteins
Fluorescent proteins such as GFP have enabled previously impossible direct biological observations. In particular, they have permitted examination of protein-protein interactions and of protein localization within cellular compartments. Modified versions of GFP offer greater flexibility such as enhanced brightness, various emission spectra and switchability.
In a chapter of the 2007 edition of Methods in Molecular Biology, Nick Thomas of GE Healthcare in Cardiff, UK, has reviewed fluorescent proteins. He points out that the advent of high-content screening has coincided with their development.
The reviewer describes how to design a high-content screening assay with fluorescent proteins and suggests criteria for selecting a target protein, a cell line and a fluorescent protein. He explains structural considerations for generating a fusion protein consisting of the fluorescent and the target proteins. He also gives examples of promoters commonly used in protein expression vectors.
The author notes that fluorescent fusion proteins can disturb normal cellular activity and, at high expression levels, fluorescent proteins can be toxic to cells. However, he states that, with appropriate cell engineering, fluorescent proteins perturb cellular function only minimally.
The reviewer observes that genetically encoded labels have been developed as alternatives to expressed fluorescent proteins. He believes that they have not yet proved their worth, although they offer a different method of labeling cells. Currently, they involve additional washing and labeling steps, and they last a finite period of time, whereas expressed fluorescent proteins are constantly replenished by the host cell.
Lastly, the author notes that most high-content screening experiments have employed stable cell lines, but creating them takes a long time. Viral expression systems promise to reduce the time.
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