Selectable Markers
By Mukta Khanolkar | 30 September 2024
Once cells are transformed by inserting the gene of interest, they need to be separated for further procedures. To do so, the cloning vector is designed to contain certain genes. These genes, called ‘selectable markers’, often give transformed cells the ability to survive in conditions where non-transformants (cells which have not taken up the plasmid) or non-recombinants (cells which have a self-ligated plasmid, which does not contain the gene of interest) struggle to grow. For example, resistance genes make transformants resistant to antibiotics, herbicides and other toxic agents.
Separating transformed cells on the basis of this newly acquired ability is called ‘positive selection’. Since untransformed cells did not integrate selectable markers into their DNA, they are rid of and this is called ‘negative selection’.
Some of the most popular selectable markers are antibiotic resistance genes. Bacteria like Escherichia coli have always been favored for biotechnological uses. Hence, it is no surprise that antibiotic resistance is used to identify transformants from non-transformants. The NPT-II gene (neomycin phosphotransferase II) confers resistance to bacteria against the antibiotics kanamycin and neomycin.
How exactly does this process of selection of transformants work? Consider the famous pBR322 plasmid, designed by Bolivar and Rodriguez (hence the name pBR322). It contains ampicillin and tetracycline resistance genes. If the gene of interest is inserted in the region of, say, tetracycline resistance, the recombinants will only be resistant to ampicillin - because the tetracycline resistance gene gets interrupted. This is called insertional inactivation.
In the antibiotic-based screening approach, the recombinants are grown on an ampicillin-containing master plate. A replica of the colony is created using a nitrocellulose membrane. This is transferred to a medium containing both ampicillin and tetracycline. This process is called replica plating. Only the non-recombinants grow (as the recombinants have lost tetracycline resistance, and the non-transformants do not have ampicillin resistance as they lack pBR322).
Thus, the transformed colonies can be selected by comparing the master and replica plates.
Fun fact! It would be a concern if these modified organisms transferred this resistance to normal cells. Hence, selectable markers are sometimes removed in subsequent generations by a process named ‘excision’.
References:
- Screening Methods in Recombinant DNA Technology; Danaher Life Sciences Library
- Genetically Modified Food/Organism: M. Rabiei, S. Sardari; Encyclopedia of Toxicology (Third Edition), 2014
- Bt Crops; Mahmood-ur-Rahman et al.; Emerging Technologies and Management of Crop Stress Tolerance, Volume 1, 2014
- Breeding Genetics and Biotechnology; H.D. Jones; Encyclopedia of Applied Plant Sciences (Second Edition), 2017
- Tissue Culture and Genetic Transformation in Sorghum bicolor; D. Balakrishna et al.; Breeding Sorghum for Diverse End Uses, 2019