Abstract: Objective To construct miR-223 knockdown lentivirus vector and provide a tool for further study of the function of miR-223. Methods According to the Invitrogen miR-RNAi online design tool, a pair of complementary oligonucleotides encoding miR-223 mature sequence was designed, annealed and ligated with pcDNA6.2-GW/EmGFP-miR vector. Then miR-RNAi expression cassette was cut and subcloned into lentiviral pCDH-CMV-MCS-EF1-copGFP vector. The lentiviruses were packaged and titered, and then ST2 cells were infected with viruses. The efficiency of infection was calculated, and the knockdown of endogenous miR-223 was detected by using real-time RT-PCR. Results Restriction enzyme digestion and sequencing results showed that miR- 223 lentivirus construct was successfully made. Lentivirus that knockdown miR-223 expression packaged and infected of target cells. The expression of GFP green fluorescent protein accounted for 80%-90% and the virus titer was 1×109 PFU/mL. The infection efficiency reached 90%. Compared with negative control virus, miR-223 knockdown lentivirus significantly down-regulated the expression of miR-223 in ST2, and was 31% (n=3, t=15.091, P＜0.05). Conclusion miR-223 knockdown lentivirus is successfully made. It provides a tool for further studying the function of miR-223.

Key words: microRNAs, lentivirus, RNA interference, lentivirus based vector, miR-223