G418 Sulfate, also known as Geneticin, is an aminoglycoside antibiotic with a structure similar to Gentamycin B1. It interferes with protein synthesis by affecting the function of the 80S ribosome and blocking the elongation step. It is toxic to prokaryotic and eukaryotic cells, including bacteria, yeast, higher plants and mammalian cells, as well as protozoa and worms. Its resistance gene (mainly the neo gene) is located in the transposon Tn601 (903) or Tn5 (derived from bacteria), but can be expressed in eukaryotic cells. These resistance genes are introduced into cells through genetic recombination technology to make them resistant to G418, so as to be used to screen and maintain the culture of prokaryotic or eukaryotic cells carrying resistance genes.
Figure 1. Chemical structures of G418 and gentamicin. (Shulman E, et al.; 2014)
In mammalian cells, when the resistance gene neo is integrated into the eukaryotic cell genome, it encodes the expression of aminoglycoside phosphotransferase (APH(3')II). This enzyme inhibits the antibiotic-ribosome interaction by covalently modifying the amino or hydroxyl functions of G418, thereby inactivating the antibiotic. This property confers resistance to cells. For stable cell line screening experiments, it is necessary to establish a killing curve (dose-response curve) to determine the minimum effective concentration for killing non-resistant cells.
In plant cells, resistance is incubated by transfecting a resistance plasmid carrying the nptII gene. The nptII gene also encodes the expression of aminoglycoside phosphotransferase, which inactivates a variety of antibiotics, including G418, kanamycin, and paromomycin.
G418 Sulfate Usage
Preparation of G418 stock solution (50 mg/mL, active concentration)
- Conversion of Activity Units
G418 units can be converted according to (1000/A0)XA1=A2, where A0 is the potency of G418, which varies from batch to batch. You can see the quality inspection report corresponding to the batch, or the label on the bottle. A1 is the active G418 concentration you want to prepare. A2 is the actual weighed powder to volume ratio concentration. For example, if the G418 activity value of the batch used is: 750 U/mg, to prepare a G418 activity concentration of 50 mg/mL, the actual powder concentration to be prepared is 1000/750×50 mg/mL=66.67 mg/mL. If you prepare 10 mL of G418 stock solution (active concentration, 50 mg/mL), you need to weigh 666.7 mg of powder.
- Sterilization and storage
According to the actual powder weight obtained by the above conversion, add 10 mL of sterile deionized water to completely dissolve it. Pre-wet the 0.22 μm syringe filter with 5 mL of sterile deionized water to remove all water. Then use this filter to filter, sterilize and divide into small amounts for single use (such as 1 mL) and freeze at -20°C. It is stable for 1 year.
Note: a) Do not filter turbid solutions, because turbid solutions mean that they are not completely dissolved, and the filtration process will cause drug loss and reduce the activity of the final solution. b) It is not recommended to use liquid culture medium, NaCl, phosphate solution or organic solvent to prepare storage solution.
Commonly used screening concentrations
Generally speaking, a high concentration of G418 is required for the initial screening of transformants, and a lower concentration of G418 is used to maintain the culture. Growth conditions, cell types and other environmental factors may affect the amount of G418 used, so it is recommended to determine the optimal screening concentration through a killing curve, i.e., a dose-response curve, for the experimental system used for the first time. Usually, the screening range for mammalian cells is 200-2000 μg/mL; plant cells: 10-100 μg/mL; yeast cells: 500-1000 μg/mL.
The following are specific reference concentrations for G418 screening of some cell types:
| Cell Types | Activation concentration | Application |
|---|
| Dictyostelium | a) 10 μg/mL
b) 30 μg/mL | a) Cultivated in culture medium
b) Cultivated on freeze-dried bacteria |
| Mammalian | a) 400 -1000 μg/mL
b) 200 μg/mL | a) For screening
b) For maintaining growth |
| Plant | a) 25-50 μg/mL
b) 10 μg/mL | a) For screening
b) For maintaining growth |
| Yeast | a) 500 μg/mL
b) 125-200 μg/mL | a) For screening
b) For maintaining growth |
| Bacteria | 16 μg/mL | For screening |
Establishment of Killing Curve
In order to screen cell lines that stably express the target protein, it is necessary to determine the lowest concentration of antibiotics that can kill untransfected host cells. This can be achieved by establishing a killing curve, and at least 6 concentrations should be selected. G418 is most active when treating cells in the dividing stage, so cells need to be cultured for a period of time before adding G418.
Application of Screening of Stably Transfected Cells
1) 48 hours after transfection, use screening medium containing appropriate concentrations of G418 to pass cells (direct passaging or passaging after dilution).
Note: Antibiotics have the best killing effect when cells are in an actively dividing state. When cells are too dense, their efficiency will decrease. For better screening results, it is best to dilute cells to an abundance of no more than 25%.
2) Replace the screening medium containing drugs every 3-4 days.
3) Observe and evaluate the formation of cell clones (colonies) after 7 days of screening. Colony formation may take another week or more, depending on the host cell type, transfection and screening effect.
4) Pick and transfer 5-10 resistant clones to a 35 mm cell culture plate and continue to culture with drug-containing screening medium for 7 days.
5) Then replace with normal culture medium for culture.
References
- Shulman E, et al.; Designer aminoglycosides that selectively inhibit cytoplasmic rather than mitochondrial ribosomes show decreased ototoxicity: a strategy for the treatment of genetic diseases. J Biol Chem. 2014, 289(4):2318-30.
- Prokhorova I, et al.; Aminoglycoside interactions and impacts on the eukaryotic ribosome. Proc Natl Acad Sci USA. 2017, 114(51): E10899-E10908.
