1 The principle of action of geneticomycin

Genetomycin (Geneticin, G418) is an aminoglycoside antibiotic that inhibits protein synthesis by blocking peptide chain extension by binding to the 70S and 80S ribosomes in prokaryotic and eukaryotic cells. This mechanism is toxic to those including bacteria, yeast, plants, and mammalian cells. When the cells were successfully transfected and integrated with a neomycin resistance gene (such as the neo gene), the gene-encoded aminoglycoside phosphotransferase (APH (3) II) could covalently modify G418 to inactivate it, thereby conferring resistance to cells grown in selective medium containing G418.

In molecular genetic tests, G418 is one of the commonly used for stable transfection.

2 The application of geneomycin

2.1 Screening of Stable Transfected Cell Lines

• G418 was used to screen for cells containing resistance genes (e. g., neo genes) by inhibiting protein synthesis. The aminoglycoside phosphotransferase encoded by the resistance gene inactivates G418, which conferring cell resistance to G418. Typically, the mammalian cell screening range is 200 – 2000 μg/mL, with the most commonly used working concentration of 100 – 700 μ g / mL. Where plant cells are 10-100 μg/mL and yeast cells are 500-1000 μ g / mL, the following are the concentrations used for some cell types using G418 for reference.

Cell	Application	Activation concentration of geneomycin(μg/mL)	Works cited
Mucor species	a) Cultured in growth medium b) Cultured on lyophilized bacteria	a) 10 μg/mL b) 30 μg/mL	Hirth,et. al., Proc. Natl. Acad. Sci., v. 79, 7356-7360 (1982).
Mammalian	a) For screening b) For maintaining growth	a) 400 -1000 μg/mL b) 200 μg/mL	Canaani and Berg, Proc. Natl. Acad. Sci., v. 79, 5166-5170 (1982).
Plant	a) For screening b) For maintaining growth	a) 25-50 μg/mL b) 10 μg/mL	Ursic, et. al., Biochem. Biophys. Res. Comm., v. 101:3, 1031-1037 (1981).
Yeast	a) For screening b) For maintaining growth	a) 500 μg/mL b) 125-200 μg/mL	Jimenez and Davies, Nature, v. 287, 869-871 (1980).
Bacteria	For screening	16 μg/mL	Waitz, et. al., Antimicrob. Agents Chemother., v. 6:5, 579-581 (1974).

2.2 Gene Knockout and Gene Transfer

• In gene knockout, G418 can be used to screen cells that successfully knockout the target gene. In gene transfer, G418 facilitates the integration of foreign genes into the genome of host cells.

2.3 Resistance Selection in Cell Culture

• During cell culture, G418 is used to screen for cells resistant to the antibiotic, which often carry specific resistance genes.

3 Relevant experimental procedures for geneomycin

3.1 Preparation of G418 Stock Solution (50 mg/mL, Active Concentration)

1) Conversion of Activity Units

Convert using the formula: (1000/A0) × A1 = A2, where A0 is the potency of G418, which varies by batch. Refer to the quality control report for the corresponding batch or the label on the bottle. A1 is the desired active concentration of G418 to be prepared. A2 is the actual powder-to-volume concentration to be weighed.

For example, if the batch of G418 has a potency of 750 U/mg and you want to prepare an active concentration of 50 mg/mL, then the actual powder concentration to be prepared is 1000/750 × 50 mg/mL = 66.67 mg/mL.

If preparing 10 mL of G418 stock solution (active concentration, 50 mg/mL), then 666.7 mg of powder is required.

• Sterilization and Storage

Based on the actual powder weight obtained from the above conversion, add it to 10 mL of sterile deionized water to completely dissolve.

First, pre-wet a 0.22 μm syringe filter with 5 mL of sterile deionized water, removing all water. Then filter using this filter to sterilize and aliquot into small volumes for single use (e.g., 1 mL) and store at -20°C for 1 year stability.

3.2 Establishment of Kill Curve

1) Day one: Plate untransformed cells at 20-25% cell density in appropriate culture plates and incubate overnight at 37°C, CO₂.

2) Set a concentration gradient within a suitable range based on cell type, selecting at least 6 concentrations (G418 is most active against cells in the division phase, so cells should be cultured for a period before adding G418), for example, for mammalian cells, set 0, 50, 100, 200, 400, 800, 1000 μg/mL.

3) Day two: Remove old culture medium and replace with freshly prepared medium containing the corresponding concentration of the drug, with three replicates for each concentration.

4) Replace with fresh drug-containing medium every 3-4 days.

5) Perform live cell counts at fixed intervals (e.g., every 2 days) to determine the appropriate concentration that prevents the growth of untransfected cells. Select the lowest concentration that can kill the majority of cells within the ideal number of days (usually 7-10 days) as the working concentration for stable transfected cell line screening.

3.3 Screening of Stable Transfected Cells

• The G418 Geneticin (60220ES) provided by Yisheng Biotech has an efficacy of ≥720 U/mg, a purity of ~98%, and batch stability, with a price nearly one-third of other companies' G418 products.
• The screening medium containing the drugs was replaced every 3-4 days.
• The formation of cell clones (colonies) was observed and assessed after 7 days of selection. Colonies may take a week or more to form, depending on the host cell type, transfection, and screening efficacy.
• 5-10 resistant clones were picked and transferred to 35 mm cell culture plates and further maintained with drug-containing screening medium for 7 days.
• After changing the normal medium culture.

4 Product recommendation

The G418 Geneticin (60220ES) provided by Yeasen Biotech has an efficacy of ≥720 U/mg, a purity of ~98%, and batch stability, with a price nearly one-third of other companies' G418 products.

Specification	S*	T*	Yeasen	Yeasen advantage
Content (HPLC)	/	/	~98%	High purity
Potency (U/mg)	≥720 µg/mg	707-721 µg/mg	≥720 µg/mg	High effectiveness
Price($/5 g)	~1000	~1000	~200	Cheapness

5 Experimental Case

In E. coli, G69 and G99 were two different batches; the concentrations of genomycin were 8 mg/L, 12 mg/L and 16 mg/L, respectively, and the effective minimum concentration was 16 mg / L, which completely inhibited the growth of miscellaneous bacteria.