Other Free Encyclopedias » Medicine Encyclopedia » Genetics in Medicine - Part 3 » Marker Systems - Selectable Markers, Screenable Markers

Marker Systems - Selectable Markers

chromosome cells gene antibiotic cell

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.

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