G418 disulfate salt

Geneticin (G418) is an aminoglycoside antibiotic similar in structure to Gentamicin produced by Micromonospora rhodorangea. Geneticin is widely used for selection of eukaryotic cells, stably transfected with neomycin resistance genes (neo) as well as maintenance of the (neor) phenotype of resistance cells. Geneticin is toxic to bacteria, yeast, protozoa, helminths and mammalian cells. It can also be used for elimination of contaminating fibroblasts from mixed cultures.

Geneticin (G418) blocks polypeptide synthesis by inhibiting protein elongation by binding irreversibility to 80S ribosomes both in prokaryotic and eukaryotic cells. This activity can be inactivated by the bacterial aminoglycoside phosphotranferases APH(3')II and APH(3')I encoded by genes on transposons Tn5 and Tn601 (903) respectively. The cells acquire resistance to Geneticin (neor) as a result of transfection of neomycin resistance genes (neo) from transposon Tn5 or Tn601 and enable the cells to grow in media containing Geneticin (G418).

This selection strategy can be used on almost any cell type. The effective concentration of geneticin varies according to the cell type, medium used, growth conditions, cell's metabolic rate and position in the cell cycle. We recommend determining optimal concentrations of antibiotic required to kill your host cell line by treating the cells with several concentrations ranging from 100ug/ml to 1mg/ml. Cells can escape selection if the antibiotic is used at too low concentrations or if the plating density is too high. Ideally cell death of control cells occurs within one week after addition of antibiotic allowing colonies of resistant cells to form by 10-14 days.

The working concentration of geneticin for selection and maintenance of mammalian cell lines transfected with neo gene depend upon many factors, including the cell type, culture media, metabolic rate of cells and cell cycle phase. The concentration of geneticin to be added to the culture medium has to be determined experimentally.

It is recommended to obtain a full dose curve when-

• New lot of geneticin is used
• Geneticin is used in new cell culture system

The effective concentrations have been reported from 100µg/ml to 5mg/ml or greater. However, the recommended working concentrations for selection in a few mammalian cell lines are listed below.

The cells are transfected with plasmid containing neo gene and are incubated in a regular growth medium containing Geneticin to select for stable transfectants. Note that the cells can escape the selection if the antibiotic is used at a too low concentration.

1. Transfect the cells with a plasmid containing neo gene.
2. 48 hours after transfection add the cells in a medium containing Geneticin at appropriate concentration.
3. Replace the antibiotic containing medium every 3-4 days.
4. After 7 days of selection evaluate the cells for the formation of foci. Foci formation may require an additional week or more depending upon the host cell line and transfection efficiency.
5. Transfer and pool 5-10 resistant clones to 35mm cell culture plate and maintain on selection medium for additional 7 days.

Store at 2-8°C away from bright light. For long term storage and sterile applications, geneticin solutions should be sterilized by filtering througha membrane with porosity of 0.22microns or less. Solutions remain stable for about 12 months at 2-8°C. For long term staorage, store at -20°C.

Shelf life is 36 months. Use before expiry date given on the product label.