1 The Mechanism of Action of Blasticidin S

Blasticidin S is a nucleoside antibiotic extracted from Streptomyces griseochromogenes, which specifically inhibits protein synthesis in prokaryotic and eukaryotic cells by interfering with the formation of peptide bonds in the ribosome. Its mechanism of action includes blocking the hydrolysis of peptidyl-tRNA induced by release factors and enhancing the binding of tRNA to the large ribosomal subunit, thereby inhibiting the formation of peptide bonds to a certain extent.

Blasticidin S has a rapid and potent mode of action, and very low antibiotic concentrations can lead to rapid cell death. It has not only have a significant inhibition of protein synthesis in bacteria and eukaryotes, but also activity against fungi, nematodes and tumor cells. Pltocidin S is often used to screen transfected cells carrying bsr or BSD genes.

2 Application of Blasticidin S

2.1 Cell Culture and Screening

• Mammalian cells: blast S is often used to screen transfected cells carrying bsr or BSD genes. These resistance genes, respectively from B. cereus and Aspergillus, were able to convert murancidin into avirulent derivatives, thereby enabling selective killing of cells without carrying resistance genes, with working concentrations of Blasticidin S in mammalian cells ranging from 1 to 50 μg/mL.

• Bacterial selection: In bacterial culture, the recommended concentration of 25-100 µg/mL can quickly cause the death of cells without resistance genes.

E.coli is slightly less sensitive to Blasticidin S, but transformants are tolerant to Blasticidin S and can be screened with low salt LB medium (pH 8) in a concentration range of 50-100 μg/mL Blasticidin S, and high pH improves Blasticidin S activity.

• Plant cells: also applicable to gene transfer experiments in plant cells, bsr and BSD are commonly used as screening markers.

2.2 Research on Protein Synthesis

• Blasticidin Swas used to study the protein synthesis process at the level of peptide bond formation, especially in HEK293-T cells and HEK-D5 cells of TLR-2 constructs.

2.3 Antifungal Properties

• It also has a certain antifungal activity, which can be used for the prevention and treatment of rice blast. Its bactericidal effect is mainly to affect protein synthesis, and can inhibit spore germination, hyphal development and spore formation.

2.4 Other Applications

• Blasticidin Shas also been used to study the inhibitory effect of tumor cells.
• It also acts as a selection agent for transformed cells containing the resistance gene bls, bsr, or BSD.

3 Experimental Procedures Related to Blasticidin S (Screening of Mammalian Stable Transfectants)

1) Blasticidin S is typically used at a concentration of 10 μg/mL. Cells are transfected with plasmids carrying the bsr or BSD gene and incubated in normal growth medium containing Blasticidin S to select for stable transfectants.

2) At 48 hours post-transfection, passage the cells in fresh medium containing an appropriate concentration of Blasticidin S (Note: Antibiotics work best when cells are in an active division phase. The efficiency of antibiotics decreases if the cell density is too high. When splitting cells, the confluence should not exceed 25%).

3) Every 3-4 days, remove the spent medium and add fresh medium containing the antibiotic.

4) After 7 days, check for the formation of cell colonies. Depending on the host cell type and transfection/selection efficiency, colony formation may require an additional week or longer.

5) Transfer 5-10 resistant clones to 35 mm cell culture dishes and maintain in selective medium for 7 days. Subsequently, perform cytotoxicity assays for testing.

4 Product recommendation

Yeasen Biotech's Blasticidin S (60218ES) is available in two forms: powder (1 g) and solution (10 mg/mL).

5 Published articles with our reagents

[1] Mo J, Tan K, Dong Y, Lu W, Liu F, Mei Y, Huang H, Zhao K, Lv Z, Ye Y, Tang Y. Therapeutic targeting the oncogenic driver EWSR1::FLI1 in Ewing sarcoma through inhibition of the FACT complex. Oncogene. 2023 Jan;42(1):11-25. doi: 10.1038/s41388-022-02533-1. Epub 2022 Nov 10. PMID: 36357572. (IF:8.0)

[2] Tan K, Mo J, Li M, Dong Y, Han Y, Sun X, Ma Y, Zhu K, Wu W, Lu L, Liu J, Zhao K, Zhang L, Tang Y, Lv Z. SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma. J Exp Clin Cancer Res. 2022 Dec 20;41(1):352. doi: 10.1186/s13046-022-02563-3. PMID: 36539767; PMCID: PMC9764568.(IF:11.3)

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