Establishment of an inhibitory oligodeoxynucleotides screening system using CAL-1-L929 cells

Objective To establish a rapid screening method for the development of novel oligodeoxynucleotides (ODN) that antagonized for the development of tumor necrosis factor-α (TNF-α). Methods The CAL-1 cells were stimulated by unmethylated CpG motif containing oligonucleotides (CpG ODN) 0800 (3 μg/ml). After 48 h, the culture supernatant was collected and TNF-α secretion in the culture supernatant was assessed by an in vitro cytotoxicity assay using L929 cells (2×104/well). After 24 h, vesicular stomatitis virus (VSV) was added into the wells, and after 72 h, the A values were detected. The experimental conditions were also well optimized. Results Screening conditions were identified as final concentration of CpG ODN 0800 of 3 μg/ml, and dilution of CAL-1 supernatant of 1/5. The A values of L929 cell was 2×104/well, and the experiment time on L929 was 24 h. SAT05f was successful screened by application of the methods above. Conclusion TNF-α can be suppressed by the ODNs with special sequence. This experimental method was successfully established for development of novel Inhibitory ODNs for targeting TNF-α. Key words: Inhibitory oligodeoxynucleotides; Toll like receptor ligand; CAL-1 cells; Tumor necrosis factor-α