Marker Systems

Screenable marker systems employ a gene whose protein product is easily detectable in the cell, either because it produces a visible pigment or because it fluoresces under appropriate conditions. Visible markers rarely affect the studied trait of interest, but they provide a powerful tool for identifying transformed cells before the gene of interest can be identified in the culture. They can also identify the tissues that have (and have not) been transformed in a multicellular organism such as a plant.

Green fluorescent protein (GFP) is used as a screenable marker or a reporter gene in a variety of cells. GFP is a small protein that is isolated from jellyfish. It possesses a trio of amino acids that absorb blue light and fluoresce yellow-green light that is detectable using a fluorescence microscope or other means. Using GFP as a reporter has the enormous advantage that transgenic cells can be located noninvasively, simply by illuminating with blue light and observing the fluorescence. It is a simple protein, and it works in many different model systems (plants, mammalian cell culture, and the like) because it requires no post-translational processing of the protein to make it active. This is helpful, because processing enzymes are typically specific to each type of organism, thus limiting the usefulness of transgenes that require such modifications. In addition, whereas some reporter products are toxic to the cell, GFP is not, and the intensity of the fluoresced light can be used to quantify gene expression.

The Escherichia coli bacterium provides another reporter gene system commonly used in plants. The bacterium makes an enzyme, called B-glucuronidase gus A (uid A), that cleaves a group of sugars called B-glucouronides. This enzyme will also cleave a chemical that is added to the culture such that the cleaved chemical is converted into an insoluble, visible blue precipitate at the site of enzyme activity. Many plants lack their own B-glucuronidase enzymes, so it is easy to determine if the plant has been transformed. Enzyme activity can be easily, sensitively and cheaply assayed in vitro, and can also be examined in tissues to identify transformed cells and tissues. The level of gene expression can be measured by the intensity of the blue color produced.