Marker Systems

Marker systems are tools for studying the transfer of genes into an experimental organism. In gene transfer studies, a foreign gene, called a trans-gene, is placed into an organism, in a process called transformation. A common problem for researchers is to determine quickly and easily if the target cells of the organism have actually taken up the transgene. A marker allows the researcher to determine whether the transgene has been transferred, where it is located, and when it is expressed (used to make protein).

The marker itself is also a gene. It is placed next to the transgene to make a single piece of DNA, which is then transferred. Markers are chosen because their gene products (proteins) have obvious effects on the pheno-type of the organism. If the system is constructed properly, detection of the marker's product indicates that the transgene is present and functioning.

Marker systems exist in two broad categories: selectable markers and screenable markers. Selectable markers are typically genes for antibiotic resistance, which give the transformed organism (usually a single cell) the ability to live in the presence of an antibiotic. Screenable markers, also called reporter genes, typically cause a color change or other visible change in the tissue of the transformed organism. This allows the investigator to quickly screen a large group of cells for the ones that have been transformed. Selectable and screenable markers are essential to genetic engineering in both prokaryotes and eukaryotes , and are often built into engineered DNA plasmids used for genetic transformation.

Selectable Markers

Selectable markers are said to cause either negative or positive selection. Negative selection kills cells that do not have the marker gene, while positive selection kills those that have it but not in the correct place in the chromosome.

Negative selection is most commonly used in the transformation of bacterial cells. A gene for resistance to an antibiotic such as kanamycin is placed on a plasmid with the transgene (such as an insulin gene). Resistance genes often code for an enzyme that phosphorylates (adds a phosphate to) the antibiotic, thereby inactivating it. Cells that take up the plasmid can thus tolerate an otherwise lethal exposure to the antibiotic. The researcher exposes the entire group of cells, and harvests those that remain alive.

Positive selection is often performed in mammalian cells grown in cell culture. Because of the complexity of the mammalian cells, it is important that a transgene not only enter the cell, but also be integrated into the correct place in the chromosome. If it does not, it is unlikely to be regulated properly. The "correct place" is the site on the chromosome where the normal gene is found. For example, if the researcher is inserting a human nerve cell gene into a mouse, it should be inserted at the site where the corresponding mouse nerve cell gene sits. Selection of cells with the properly located transgene is accomplished by killing off transformed cells in which the gene is in the wrong place.

This system, an example of positive selection system, has three parts. The first is an antibiotic, the second is an enzyme that acts on the antibiotic, and the third is an enzyme that cuts and splices DNA.

The antibiotic ganciclovir is used to kill cells. Ganciclovir is a "nucleotide analog," meaning it is structurally similar (but not identical) to the building blocks of DNA. It must be phosphorylated before it can be incorporated into DNA in the target cell. Once it is incorporated, it acts like a monkey wrench in the machinery, preventing normal DNA function and thus killing the cell. The enzyme that acts on the ganciclovir is called thymidine kinase (TK). It adds a phosphate on the antibiotic, inactivating the antibiotic. Mammalian TK does not phosphorylate ganciclovir very efficiently, so mammalian cells are not normally killed by it. TK from the Herpes simplex virus (HSV) does phosphorylate it efficiently, and any mammalian cell transformed with an active HSV TK enzyme will be killed.

In this system, a plasmid is constructed with the transgene , the HSV TK gene, and a "recombination site," a stretch of DNA that is recognized by the cellular recombinase enzymes that cut and splice DNA. If the trans-gene is integrated into the chromosome at the site of the normal gene, then the HSV TK gene is eliminated by the cellular "recombinase" enzymes, and the cells are not sensitive to ganciclovir. In improperly transformed cells, the recombinase can't remove the HSV TK gene, and so those cells will be killed when exposed to ganciclovir.

Screenable Markers

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.

see also Cloning Genes; Model Organisms; Plasmid; Post-translational Control; Recombinant DNA.

Linnea Fletcher

Bibliography

Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.

Ponder, Bruce A. "Cancer Genetics." Nature 411 (2001): 336-341.

Risch, Neil J. "Searching for Genetic Determinants in the New Millenium." Nature 405 (2001): 847-856.