Transfection of mammalian cells with fluorescent protein fusions

Fluorescent protein fusions (FPFs) have been used to address a wide range of questions in individual cells as well as in specific tissues of particular organisms. However, investigators must take extreme care when using FPFs to ensure that the resultant fusion protein is expressed at or close to the endogenous level of the parent protein, and also that it is full length, localizes correctly, and behaves normally once incorporated in the cell. Although transient transfection methods can be used to introduce DNA coding for FPFs, in many cases it is beneficial and/or essential to develop stable cell lines expressing the fusion protein of interest. In addition to providing more native levels of expression, the individual clones can be generated from single cells, the integration site of the plasmid can be mapped, and the copy number can be determined. Moreover, because every cell in the population is expressing the fusion protein, cell cycle analyses and biochemical fractionation are significantly easier to accomplish. This article presents a protocol for generating, selecting, and screening stable cell lines expressing FPFs.