G418 (geneticin) is an

aminoglycoside Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside (sugar). The term can also refer ...

antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...

similar in structure to gentamicin B1. It is produced by '' Micromonospora rhodorangea''. G418 blocks

polypeptide synthesis Protein biosynthesis, or protein synthesis, is a core biological process, occurring inside cells, balancing the loss of cellular proteins (via degradation or export) through the production of new proteins. Proteins perform a number of critical ...

by inhibiting the elongation step in both

prokaryotic A prokaryote (; less commonly spelled procaryote) is a single-celled organism whose cell lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Greek (), meaning 'before', and (), meaning 'nut' ...

and

eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...

cells. Resistance to G418 is conferred by the neo gene from Tn5 encoding an aminoglycoside 3'-phosphotransferase, APT 3' II. G418 is an analog of

neomycin Neomycin, also known as framycetin, is an aminoglycoside antibiotic that displays bactericidal activity against Gram-negative aerobic bacilli and some anaerobic bacilli where resistance has not yet arisen. It is generally not effective against ...

sulfate, and has similar mechanism as

. G418 is commonly used in laboratory research to select genetically engineered cells . In general, for bacteria and algae, concentrations of 5 μg/mL or less are used; for mammalian cells, concentrations of approximately 400 μg/mL are used for selection and 200 μg/mL for maintenance. However, optimal concentration for resistant clones selection in mammalian cells depends on the

cell line An immortalised cell line is a population of cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...

used as well as on the

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...

carrying the resistance gene. Therefore, antibiotic titration should be done to find the best condition for every experimental system. Titration should be done using antibiotic concentrations ranging from 100 μg/mL up to 1400 μg/mL. Resistant clones selection could require from 1 to up to 3 weeks. __TOC__

G418 impurity profile

G418 is produced by

fermentation Fermentation is a type of anaerobic metabolism which harnesses the redox potential of the reactants to make adenosine triphosphate (ATP) and organic end products. Organic molecules, such as glucose or other sugars, are catabolized and reduce ...

and isolation processes and the G418 producing strain '' Micromonospora rhodorangea'' produces many other gentamicins while producing G418. The common impurities of G418 include gentamicins A, C1, C1a, C2, C2a and X2. The quality of G418 is not based on just the potency, but more on the selectivity defined by the killing curve of the sensitive cells vs the resistant cells. A good G418 product has the lowest for sensitive cells (such as NIH 3T3) and the highest LD₅₀ (can be up to 5,000 μg/ml) for resistant cells (NIH 3T3 transfected with resistant genes). Gentamicins have almost no selectivity, except gentamicin X2.

Use in cell biology

G418 is routinely used as a selective agent in cell culture set-ups. Researchers can link the ''neoR'' selective resistance gene with their vector. Then if the vector is successfully introduced into cells, the cells can become G418-resistant cells. After treating with G418, these vector(-) cells will die, while vector(+) cells will survive. This method can help researchers select vector(+) cells.

Mechanism of action

G418 Disulfate and other aminoglycosides prevent protein synthesis at the early stages of elongation, post-initiation, initiation of translation. Resistance to G418 Disulfate is conferred by the Neomycin resistance gene (''neo)'' from either Tn5 or Tn601 (903) transposons. Cells transfected with resistance plasmids containing the ''neo'' gene can express aminoglycoside 3'-phosphotransferase (APT 3' I or APT 3' II) which covalently modifies G418 to 3-phosphoric G418, which has negligible potency and has low-affinity for prokaryotic and eukaryotic ribosomes.

References

{{reflist Aminoglycoside antibiotics Cell culture reagents Eukaryotic selection compounds