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Description

SimpliconMBOL> is a novel system to effect immediate high sustained protein expression of multiple genes into transfected cells without the risk of genome integration. The technology employs a single, synthetic, polycistronic, self-replicating RNA based on the Venezuelan equine encephalitis (VEE) genome1,2,3,4. The SimpliconMBOL> RNA contains only genes encoding the VEE RNA replication machinery while the structural proteins that are required to make an infectious particle have been removed and replaced with the transgenes of interest. The SimpliconMBOL> RNA is a synthetic RNA generated from the SimpliconTM Cloning Vector (E3L) plasmid.

Introduction and replication of the SimpliconMBOL> RNA is expected to elicit a strong interferon response in transfected cells. To suppress the IFN responses, a Vaccinia virus protein5, B18R, is used for the original SimpliconTM technology.

One day after transfection of the SimpliconMBOL> RNA, a spike in the levels of transgenes can be observed. The expression levels are maintained by addition of B18R, E3L and the selective agent, puromycin throughout the duration of the experiment. Over time, expression levels are expected to diminish and stabilize to 1/5 1/10 the levels initially observed and may be close to physiological levels after one week. Expression levels and duration may change depending upon the cell types, transgenes and media conditions used. The SimpliconMBOL> technology has been successfully utilized for efficient human iPSC generation through the sustained expression of critical reprogramming factors3,4 and in the creation of cell lines that express and retain the metabolic activities of five cytochrome P450 enzymes6.

In the SimpliconMBOL> Expression System, B18R is provided as a B18R RNA (Cat. No. SCR722) for the suppression of IFN responses at RNA transfection. For sustained transgene expression, recombinant B18R protein (Cat. No. SCR156 and SCR197) or B18R conditioned medium (B18R-CM) can be used. B18R-CM can be produced from B18R RNA synthesized using B18R plasmid (Cat. No. SCR728), respectively.

The B18R Plasmid (human codon optimized) (Cat. No. SCR728) is used as a DNA template for the synthesis of B18R RNA (Cat. No. SCR722). The B18R RNA is a synthetic mRNA and is used for co-transfected with the SimpliconMBOL> RNA. B18R RNA can be used to prepare B18R-CM by transfection into HFFs (Cat. No. SCC058). B18R RNA is also available for co-transfection of any kinds of mRNA to suppress the IFN responses. Please refer to the User Guide for SimpliconMBOL> Expression System located on our website for detailed protocols.