Puromycin dihydrochloride (Synonyms: CL13900)

Cell lines

Fetal porcine somatic cells

Preparation Method

Cells were seeded in 24-well plates at a density of 2.5 x 10⁴ cells per well and cultured in medium containing 0.5–6 μg/ml puromycin dihydrochloride. Stock solution (10 mg/ml) of puromycin dihydrochloride was prepared by dissolving puromycin dihydrochloride in distilled water at the appropriate concentration. Media containing variable amounts of puromycin dihydrochloride were freshly prepared by adding the appropriate volume of puromycin dihydrochloride stock solution.

Reaction Conditions

0.5–6 μg/ml for 7 d

Applications

Puromycin dihydrochloride is an antibiotic that inhibits growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses. To determine the optimal concentration of puromycin dihydrochloride for selecting EGFPac-transfected cells, a puromycin dihydrochloride resistance test was performed with fetal porcine somatic cells. The puromycin-resistant gene (termed pac) encoding puromycin N-acetyl transferase was isolated from Streptomyces aboniger. If pac is introduced and expressed in animal cells, the cells can survive in the presence of puromycin dihydrochloride.

Animal models

Female FVB/N mice, 8–10 weeks old.

Preparation Method

Puromycin dihydrochloride was dissolved in 100 μl of PBS. Mice were housed under a 12-h light/dark cycle with ad libitum access to food and water unless otherwise stated. Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) before all surgical procedures.

Dosage form

Puromycin dihydrochloride was intraperitoneal injected to mice with a concentration of 0.040 μmol/g.

Applications

The antibiotic puromycin dihydrochloride (a structural analog of tyrosyl-tRNA), and anti-puromycin antibodies could be used to detect the amount of puromycin incorporation into nascent peptide chains as well as to measure changes in protein synthesis in cell cultures.

References:

[1]. Watanabe S, Iwamoto M, et al. A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin. Biol Reprod. 2005 Feb;72(2):309-15.

[2]. Goodman CA, Mabrey DM, et al. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 2011 Mar;25(3):1028-39.