Abstract:

BACKGROUND: Aseptic loosening of the prosthesis has become the most common complication of total joint replacement and reduces prosthesis lifespan. Tumor necrosis factor-α (TNF-α) is a novel target for treating aseptic loosening. Small interfering RNA (siRNA) as a novel gene blocking technique has been widely used in gene screening, identification, signal transduction and gene therapy.
OBJECTIVE: To explore the blocking effect of siRNA on the expression of TNF-α gene in U937 cell line using siRNA eukaryotic expression vector.
METHODS: Two TNF-α siRNA cDNAs were synthesized according to the TNF-α gene sequence and cloned into the vector pSilencer4.1-CMV-neo and named pSilencer-T1 and pSilencer-T2, respectively, which were further identified by restriction endonuclease digestion analysis and DNA sequencing. U937 cells were transfected with pSilencer-T1 and pSilencer-T2 by electrotransfer. After G418 selection, the cells were selected, and the interfering effect was detected by RT-PCR.
RESULTS AND CONCLUSION: Restriction endonuclease digestion analysis and DNA sequencing results showed that the target segments were cloned into pSilencer4. 1-CMV neo vector respectively. The results of RT-PCR indicated that both siRNA vectors could successfully knock down TNF-α gene expression. The vector-based siRNA on TNF-α gene can effectively knock down TNF-α gene expression. Results showed that pSilencer-T1 and pSilencer-T2, human TNF-α gene siRNA eukaryotic expression vector were successfully constructed and blocked TNF-α gene expression.