Commonly know as G418 or G-418, Geneticin® reagent is an aminoglycoside related to Gentamicin and is commonly used as a selective agent for eukaryotic cells.  As an analog of neomycin sulfate, it interferes with the function of 80S ribosomes and protein synthesis in eukaryotic cells.  The dominant-acting resistance genes (neor) used on common mammalian expression vectors contain elements derived from either transposons Tn601 (903) or Tn5.  When grown in medium containing Geneticin® selection agent, stable colonies of mammalian cells expressing these resistance markers can be generated in 10 to 14 days.

What's in your G-418?

To be effective, selection agents need to be reliable, highly pure, and consistent in nature.  Geneticin® selective antibiotic, considered the gold standard for nearly two decades, is the only G-418 reagent that delivers these qualities to your mammalian cell cultures.  Compared to other manufacturers’ G-418 products, Geneticin® antibiotic exhibits higher purity so you can use less and produce greater selection pressure with minimal toxicity from contaminants.

When evaluating G-418 products, consider more than just potency.  Look at purity and working range.  Taken together, these three characteristics provide a true evaluation of the effectiveness of your G-418.
Purity.  The purity of Geneticin® selection agent is consistently greater than 90%, as determined by HPLC; far higher than any other supplier. This means:

  • 15-30% lower concentrations of Geneticin® agent can be used as compared to other G-418 products to achieve comparable selection results.
  • Surviving clonal colonies may arise faster and cells may appear healthier when using Geneticin® instead of lower purity G-418 products.
Potency.  Potency as a descriptive term is often used synonymously with purity, but potency as reported in QC data is actually not related to purity.  The potency value is a measure of bacterial growth inhibition.

  • Gentamicins that commonly contaminate G-418 preparations from other suppliers will add to potency in bacterial assays yet have no effect on mammalian cell selectivity.
  • Other bacterial contaminants in impure G-418 may be toxic to mammalian cells, consequently lowering the ED 50 value.

Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
ED50 Assay .  This is a true measure of eukaryotic growth selectivity as determined on reference NIH3T3 cells.  Higher purity generally translates to higher ED50 values.

  •  Toxic impurities lower the ED50 value, which means a more narrow working range of antibiotic selection.
  •  A consistent ED50 from a supplier assures performance reproducibility from lot-to-lot in mammalian cell culture applications.

This means when purchasing a new lot of Geneticin®, you do not need to re-optimize for ideal antibiotic concentration (assuming that no other media alterations).  The wide ranges of ED50 displayed in different lots from alternative G-418 suppliers may require you to re-optimize for each lot.  Lot-to-lot consistency with Geneticin® is sufficiently high that you will not notice any difference in selection performance between lots.

Table 1. Comparison of Geneticin® specifications with alternative G-418 suppliers.

Specification
Invitrogen
Supplier A
Supplier B
The Geneticin® Advantage
Purity (HPLC)
>90-93%
66-75%    65-82%Significantly higher purity
Potency claimed (µg/mg)
718-735
712-724
673-735
More stringent potency assay
Potency upon retest (µg/mg)
718-735
640-659
621-677
Consistently higher actual potency
ED50 Assay (µg/ml)
2450-2700
1350-3100
600-2350
Reliable selection lot-to-lot

Table 1. Multiple lots of G-418 from two different suppliers were analyzed according to Invitrogen raw material QC protocols for Geneticin®.  It was observed that results obtained with G-418 from Supplier A and Supplier B displayed wide variations on all measured parameters, including an inconsistency between the actual potency and the value reported on the supplied product literature.  Discrepancies between re-tested potency values and that claimed by other suppliers may be due to differences in G-418 reference lots and assay procedures.

Figure 1. Geneticin® displays greater purity than G-418 from alternative suppliers.



Figure 1. Comparison of HPLC data of Geneticin® with selected lots of G-418 from Supplier A and Supplier B.  Samples were prepared and analyzed according to the procedure published in Clinical Chem. (1978), Vol. 24, No.11, p1940.