Establishment of miR-223 knockdown lentivirus / 天津医药

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 oligo?nucleotides encoding miR-223 mature sequence was designed, annealed and ligated with pcDNA6.2-GW/EmGFP-miR vec?tor. 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 calcu?lated, 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 knock?down 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.