Abstract
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 (Sf9) 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 Sf9 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.
Highlights
The baculovirus-insect cell expression system (BEVS) is widely used for the manufacturing of various recombinant products, e.g., virus-like particles to be applied as vaccines [1] or display scaffolds [2], viral vectors for gene therapy [3], gene delivery vectors [4], and various other complex proteins
Our approach is based on combining the transcription machinery of the prokaryotic bacteriophage T7 [23] with RNA interference (RNAi)-based artificial microRNA (amiRNA) constructs derived from the AcMNPV-pri-miR-1 transcript [20]
Notwithstanding that a slightly different enhanced green fluorescent protein (eGFP) variant was applied in this study, the synthetic duplex embedded in the amiRNA constructs was still applicable to estimate the silencing capacity of the proposed system, as the enhanced yellow fluorescent protein (eYFP) sequence used in these experiments contained the target nucleotides of the published synthetic small-interfering RNAs (siRNAs)
Summary
The baculovirus-insect cell expression system (BEVS) is widely used for the manufacturing of various recombinant products, e.g., virus-like particles to be applied as vaccines [1] or display scaffolds [2], viral vectors for gene therapy [3], gene delivery vectors [4], and various other complex proteins. The popularity of this platform lies in its advantageous properties that allow for high product titers in combination with lower production costs as compared to production in mammalian cells [5] as well as proper post-translational modifications such as glycosylation [6]. Unlike siRNAs, miRNAs show imperfect complementarity to their target mRNA sequences, and they mediate translational repression and transcript degradation [9]
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