Puromycin

Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger and inhibits protein synthesis by binding to RNA. Considering that the fact part of puromycin molecule resembles the 3′ end of the aminoacylated tRNA, puromycin can enter the A site of ribosomes and transfer into the growing chain, causing premature chain release during translation in the ribosomes, and thus blocking protein synthesis.

Fig.1 Puromycin Chemical Structure (CAS NO. 53-79-2)

The pac gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger endows cells with resistance to puromycin. Exogenous expression of the pac gene is commonly used in conjunction with puromycin to generate stable cell lines. The widespread application of puromycin in the screening of stable cell lines is related to the characteristics of commercial lentiviral vectors, most of which carry pac gene. In certain conditions, puromycin can also be used to screen E.coli strains carrying pac gene. The recommended working concentration ranges from 2-5.0 µg/mL for adherent mammalian cell, but it can be toxic to suspension cells at concentrations as low as 0.5-2 µg/mL. Mammalian cells stably resistant to puromycin can be established within one week.

Establish Stable Cell lines

Experimental principle

The exogenous DNA/shRNA can be mobilized on a plasmid carrying pac gene and used to transfect a host cell in an attempt to integrate into the host genome, so that foreign genes can be expressed in the host cells for a long time. The resistance markers, pac gene, contained in the vector were used for screening, therefore puromycin can be used in gene selections for mammalian host cells.

Preparation

1. Puromycin recommending working concentration.

Mammalian cells: 1-10 μg/mL.

E.coli: 125 μg/mL in LB medium. Accurate pH adjustment is required.

2.  Puromycin dissolution

To prepare a stock solution, dissolve puromycin with distilled deionized water into 50 mg/mL solution. Filter and sterilize with 0.22 μm membrane filter. Aliquot the solution and store at -20℃. Avoid repeated freezing and thawing.

3. Puromycin kill curve (For shRNA or lentivirus transduction)

The working concentration of puromycin varies with cell type, media, growth conditions and cell metabolic rate. Before stable transfected cell lines can be selected, the optimal puromycin concentration needs to be determined by performing a kill curve titration.

1) Seed 5~8×104 cells in each well of 24-well culture plate and incubate the cells at 37°C overnight.

2) Replace the medium with fresh medium containing various concentrations of Puromycin (at least 5 continuous concentration gradient) and incubate at 37°C.

3) Refresh the selective medium every 2-3 days and observe the percentage of surviving cells over time.

4) Determine the lowest concentration of antibiotic that kills 99% of the non-resistant cells within 4-6 days.

Stable cell lines Screening

Generally, a high concentration is required initially for screening transforters, and a low concentration to maintain cell culture.

1) 48 h after transfection, replace the medium with culture medium containing appropriate concentration of puromycin.

2) Perform regular fluid changes and monitor the growth of the cells. To achieve a stable cell pool, the antibiotic selection should be sustained at least as long as it takes the control (untransduced) cells to completely die.

3) After that, the cells may undergo additional selection while the population expands. Once the polyclonal populations are growing well and have been sufficiently expanded, prepare cell stocks and/or harvest to test for protein expression.

[Note] ① Antibiotics work best on actively dividing cells. If the cells are too dense, the antibiotic will not kill the cells. Split the cells such that the cells are no more than 25% confluent. ② Monitor the growth of the cells everyday. The screening cycle of puromycin with effective concentration is generally 3-10 days. ③ Given the MOI of the lentivirus used, cells may also exhibit varying copies of pac gene, resulting in varying tolerance to puromycin. Therefore, the concentration of puromycin needs to be adapted, but it could not be lower than the minimum effective concentration of the kill curve. 

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Citations & References (Incomplete statistics)

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