Selectable marker explained

Selectable markers are a genes introduced into a cell, especially a bacterium or to cells in culture, that confer a traits suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes: bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered useful selectable markers for E. coli.

Modus operandi

The non-recombinants are separated from recombinants; that is, an r-DNA is introduced in bacteria, and some bacteria are successfully transformed while some remain non-transformed. When grown on a medium containing ampicillin, bacteria die due to lack of ampicillin resistance. The position is later noted on nitrocellulose paper and separated out to move them to a nutrient medium for mass production of the required product. An alternative to a selectable marker is a screenable marker, which can also be denoted as a reporter gene, which allows the researcher to distinguish between wanted and unwanted cells, such as between blue and white colonies. (see Blue–white screen) These wanted or unwanted cells are simply non-transformed cells that were unable to take up the gene during the experiment.

Positive and Negative

For molecular biology research, different types of markers may be used based on the selection sought. These include:

• Positive or selection markers are selectable markers that confer selective advantage to the host organism.^[1] An example would be antibiotic resistance, which allows the host organism to survive antibiotic selection.
• Negative or counterselectable markers are selectable markers that eliminate or inhibit growth of the host organism upon selection.^[2] An example would be thymidine kinase, which makes the host sensitive to ganciclovir selection.
• Positive and negative selectable markers can serve as both positive and negative markers by conferring an advantage to the host under one condition, but inhibiting growth under a different condition. An example would be an enzyme that can complement an auxotrophy (positive selection) and be able to convert a chemical to a toxic compound (negative selection).

Common examples

Examples of selectable markers include:

• Beta-lactamase, which confers ampicillin resistance to bacterial hosts.
• Neo gene from Tn5, which confers resistance to kanamycin in bacteria and geneticin in eukaryotic cells.^[3]
• Mutant FabI gene (mFabI) from the E. coli genome, which confers triclosan resistance to the host.^[4]
• URA3, an orotidine-5' phosphate decarboxylase from yeast, is a positive and negative selectable marker. It is required for uracil biosynthesis and can complement URA3 mutants that are auxotrophic for uracil (positive selection). The enzyme URA3 also converts 5-fluoroorotic acid (5FOA) into the toxic compound 5-fluorouracil, so any cells carrying the URA3 gene will be killed in the presence of 5FOA (negative selection).^[5]

Future developments

In the future, alternative marker technologies will need to be used more often to, at the least, assuage concerns about their persistence into the final product. It is also possible that markers will be replaced entirely by future techniques which use removable markers, and others which do not use markers at all, instead relying on co-transformation, homologous recombination, and recombinase-mediated excision.^[6]

See also

• Genetic marker
• Marker gene
• Biomarker

Notes and References

1. Web site: positive selection. Scitable. Nature. 29 September 2011.
2. Web site: negative selection. Scitable. Nature. 29 September 2011.
3. http://www.cellmigration.org/resource/komouse/images/mousefig2.png Callmigration.org: Gene targeting
4. Jang. Chuan-Wei. Magnuson, Terry . A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli. PLOS ONE. 20 February 2013. 8. 2. e57075. 10.1371/journal.pone.0057075. 23437314. 3577784. 2013PLoSO...857075J. free.
5. Boeke JD . LaCroute F . Fink GR . A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance . Mol. Gen. Genet. . 197 . 2 . 345–6 . 1984 . 6394957 . 10.1007/bf00330984. 28881589 .
6. Goldstein . Daniel A. . Tinland . Bruno . Gilbertson . Lawrence A. . Staub . J.M. . Bannon . G.A. . Goodman . R.E. . McCoy . R.L. . Silvanovich . A. . Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies . . Society for Applied Microbiology (Wiley) . 99 . 1 . 2005 . 1364-5072 . 10.1111/j.1365-2672.2005.02595.x . 7–23. 15960661 . 40454719 .