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[http://www.openbiosystems.com/ Open Biosystems]
[http://www.openbiosystems.com/ Open Biosystems]
Revision as of 18:38, 30 October 2008
Puromycin dihydrochloride is a aminonuclease antibiotic that inhibits protein synthesis. Puromycin is used for selection and maintenance of cell lines expressing a transfected pac gene (S. alboniger), whose product, puromycin acetyltransferase, inactivates puromycin via acetylation; recommended concentration in cell culture 1-10µg/ml. Lot-to-lot variations in potency exist for all selection antibiotics, each new lot of puromycin should be titrated. The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.
Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition. Media should be changed out every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.
Puromycin Selection (shRNA protocol) for selection of stably transfected cells
- 48 hours post-shRNA plasmid transfection, aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
- Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.
- Monitor the cells daily. Puromycin selection requires a minimum of 48 hours.
- Optimum effectiveness should be reached within 3-10 days.
- Assay transfected cells.
Suggested working conditions for selection in some mammalian cells:
- Hela human uterus: 3 µg/ml
- HEK293 human embryonic kidney: 3
- B16 mouse melanoma: 1-3
- PC1.0 hamster adenocarcinoma: 10
Titrating puromycin for selecting transfected cell lines
- Plate 2 x 105 cells in each well of a 6-well plate containing 3 ml of the appropriate complete medium plus increasing concentrations of puromycin (i.e., 0, 1.0, 2.5, 5.0, 7.5, and 10.0 µg/ml)
- Replace with fresh selective medium after 2 days to remove dead cells.
- Examine the wells for viable cells every two days.
- Monitor the cells daily and observe the percentage of surviving cells. Optimum effectiveness should be reached in 1-4 days.
- The minimum antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 3-5 days from the start of puromycin selection.
Factors Influencing Successful Transfection
1. Concentration and purity of nucleic acids – Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.
2. Transfection in serum-free media – the highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.
3. No antibiotics in transfection medium – The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.
4. High protein expression levels – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.
5. Cell history, density, and passage number–It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.
Murnane, J. P., Yezzi, M. J., Young, B. R. (1990) Recombination events during integration of transfected DNA into normal human cells. Nucleic Acids Res, 18: 2733-2738.
Haber, J. E. (1999) DNA repair. Gatekeepers of recombination. Nature, 398: 665-667.
Glover, D. J., Lipps, H. J., Jans, D. A. (2005). Towards safe, non-viral therapeutic gene expression in humans. Nature Rev Gen, 6: 299-310.