Development of a Dual-Vector System Utilizing MicroRNA Mimics of the miR-1 for an Inducible Knockdown in Insect Cells.

The baculovirus-insect cell expression system is a popular tool for the manufacturing of various attractive recombinant products. Over the years， several attempts have been made to engineer and further improve this production platform by targeting host or baculoviral genes by RNA interference. In this study， an inducible knockdown system was established in insect (9) cells by combining an artificial microRNA precursor mimic of baculoviral origin and the bacteriophage T7 transcription machinery. Four structurally different artificial precursor constructs were created and tested in a screening assay. The most efficient artificial microRNA construct resulted in a 69% reduction in the fluorescence intensity of the target enhanced yellow fluorescent protein (eYFP). Next， recombinant baculoviruses were created carrying either the selected artificial precursor mimic under the transcriptional control of the T7 promoter or solely the T7 RNA polymerase under a baculoviral promoter. Upon co-infecting 9 cells with these two viruses， the fluorescence intensity of eYFP was suppressed by ~30⁻40% on the protein level. The reduction in the target mRNA level was demonstrated with real-time quantitative PCR. The presented inducible knockdown system may serve as an important and valuable tool for basic baculovirus-insect cell research and for the improvement of production processes using this platform.