CONDITIONS OF SELECTION

Puromycin is poorly active on E. coli but is particularly useful in experiments conducted with mammalian cells. It can be used as an alternative to neomycin system for transfection experiments.

Mammalian cells :

The working concentrations of puromycin for mammalian cell lines range from 1 to 10 µg/ml. It is recommended that an initial experiment be done to determine the optimal concentration of puromycin required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene. Dying cells detach from the plates allowing easy and early identification of transformant clones. Suggested working conditions for selection in some mammalian cells are listed below:

Method (Selection procedure for mammalian cells)

Puromycin is normally used at a concentrations ranging from 1 to 10 µg/ml. After transformation with a plasmid containing the pac gene, cells are incubated in their regular growth medium containing Puromycin to select for stable transfectants.

1. 48 hours post-transfection, pass cells (direct or diluted) in fresh medium containing Puromycin at the appropriate concentration.
   Note: Antibiotics work best when cells are actively dividing. If the cells become too dense, the antibiotic efficiency will decrease. It is best to split cells such that they are not more than 25% confluent.
2. Remove and replace antibiotic containing medium every 3-4 days.
3. Evaluate cells for the formation of foci after 7 days of selection. Foci may require an additional week or more to develop depending on the host cell line and transfection/selection efficiency.
4. Transfer and pool 5-10 resistant clones to a 35mm cell culture plate and maintain on selection medium for an additional 7 days. This pooled culture will be expanded for subsequent cytotoxicity assays.

Escherichia coli :

Puromycin-resistant transformants are selected in LB agar medium supplemented with 125 µg/ml of puromycin. But use of puromycin for E. coli selection requires precise pH adjustment and also depends on which strain is selected. For hassle–free selection and optimum results the use of Fast-Media® PURO is highly recommended. Plates containing puromycin are stable for 1 month when stored at 4°C.

References

• Vara J., Perez-Gonzalez J.and A.Jimenez (1985) Biosynthesis of puromycin by Streptomyces alboniger. Characterization of puromycin N-acetyltransferase. Biochemistry 24: 8074-8081
• Lacalle R.A.,Pulido D., Vara J., Zalacain M. and A. Jimenez (1989) Molecular analysis of the pac gene encoding a puromycin-N-acetyl transferase from Streptomyces alboniger. Gene 79: 375-380
• De La Luna S. and J.Ortin (1992) Pac gene as efficient dominant marker and reporter gene in mammalian cells. Methods In Enzymology 216:376-385