Puromycin is an aminoglycoside antibiotic produced by Streptomyces alboniger. It is an analog of the 3' end of the aminoacyl-tRNA molecule and can bind to the A site of the ribosome, incorporating into the extending peptide chain, disrupting peptide translocation on the ribosome, causing premature chain termination during translation, and thus inhibiting protein synthesis.

Figure 1 Structural formula of Puromycin (CAS NO. 53-79-2)

The pac gene, encoding puromycin N-acetyltransferase (pac), was found in the Puromycin-producing bacterium Streptomyces alboniger, conferring resistance to puromycin. This characteristic is now widely used to screen mammalian stable transfected cell lines carrying plasmids with the pac gene. The widespread use of puromycin in the screening of stable cell lines is related to the characteristics of lentiviral vectors, most of which carry the pac gene in commercial lentiviral vectors. In certain specific cases, puromycin can also be used to screen E. coli strains transformed with plasmids carrying the pac gene. Puromycin acts rapidly and can quickly cause cell death at low concentrations. Adherent mammalian cells are sensitive to concentrations of 2-5 µg/mL of puromycin, while suspension cells are already sensitive to concentrations as low as 0.5-2 µg/mL of puromycin. Mammalian cells with stable resistance to puromycin can be generated within a week.

Stable Cell Line Screening

Experimental Principle

By cloning exogenous DNA/shRNA into a vector carrying the pac gene, the recombinant vector is transfected into host cells and integrated into their chromosomes, stably transmitted with cell division, and finally screened with puromycin.

Preparation and Pre-experiment

1 Recommended Usage Concentrations

Mammalian cells: 1-10 μg/mL, the optimal concentration needs to be determined by pre-experiment.

E.coli: For screening stable transformed E. coli carrying the pac gene on LB agar medium, use a concentration of 125 μg/mL. Precise pH adjustment is required for puromycin screening of E. coli stable transformants.

2 Dissolution Method

Dissolve puromycin in distilled water to prepare a 50 mg/mL stock solution, filter sterilize with a 0.22 μm filter, aliquot, and store at -20°C.

3 Determination of Puromycin Kill Curve (using shRNA transfection or lentiviral transduction as an example)

The effective screening concentration of puromycin is related to cell type, growth status, cell density, cell metabolism, and the cell cycle position. It is crucial to determine the minimum concentration of puromycin that kills untransfected cells. It is essential to perform a Puromycin gradient screening pre-experiment for the first experiment to establish a kill curve.

• Plate cells at a density of 5~8×10⁴cells/well in a 24-well plate and culture overnight.
• Prepare screening medium: Fresh medium containing different concentrations of puromycin (e.g., 0-15 μg/mL, at least 5 gradients).
• Replace with freshly prepared screening medium, continue culturing, and observe cell growth, changing the fresh screening medium every 2-3 days.
• Observe cell survival daily; the drug concentration that effectively kills all non-transfected cells within 4-6 days is the minimum concentration.

Screening of Stable Transfectants

• After lentiviral infection for 48-72h (70-80% confluence), culture cells in medium containing an appropriate concentration of Puromycin for at least 48h. When all cells in the non-transfected control group with puromycin die, the remaining cells in the virus-infected group are all positive cells.

【Note】: ① The effect of antibiotics is most pronounced when cells are in an active division phase. The efficacy of antibiotics will be significantly reduced if the cells are too dense. The cell density should not exceed 25%. ② Observe the cell growth status daily; puromycin screening requires at least 48 h, and the effective concentration of puromycin screening usually lasts from 3 to 10 days. The higher the MOI of the virus, the more copies of shRNA and puromycin resistance gene each cell contains. When performing puromycin screening, the higher the MOI, the more pac copies the cells contain, and the higher the concentration of puromycin they can tolerate. Adjust the concentration of puromycin to screen transfected cells, but the concentration of puromycin should not be lower than the minimum effective concentration of the kill curve;

• Add culture medium containing the minimum concentration of Puromycin to expand cells, and after the qPCR test results are qualified, proceed with cryopreservation.

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