TagRFP Simplicon(R) RNA (E3L) Kit

TagRFP Simplicon(R) RNA (E3L) Kit To determine optimal transfection conditions to express the self-replicating RNA of your interest through using Simplicon Cloning Vector (E3L) (Part #:SCR724) in hard-to- transfect somatic or primary cells. Simplicon 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. The Simplicon 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. Introduction and replication of the Simplicon RNA is expected to elicit a strong interferon response in transfected cells. To suppress the IFN responses, a Vaccinia virus protein4, B18R, is used for the original Simplicon technology. Recently, we found that another Vaccinia virus protein4, E3L, also suppresses the IFN responses in Simplicon RNA expression. B18R neutralizes type I interferons by direct binding, while E3L inhibits the cytoplasmic signaling pathwaycs of IFN responses. Therefore, B18R and E3L are both employed in the Simplicon Expression System and work collaboratively to suppress IFN responses. As a result, there is increased cell viability during RNA transfection and increased expression of the transgenes. The Simplicon Expression System works in human cells and is not expected to work in mouse cells. This is because the B18R does not effectively neutralize mouse interferon (IFN)-beta.The TagRFP Simplicon RNA (E3L) Kit was developed to enable the evaluation of the Simplicon Expression System in targeted cells. E3L4 incorporation in the Simplicon Expression System strongly suppresses interferon responses and enables increased expression of the target gene(s). TagRFP Simplicon RNA expresses a monomeric red (orange) fluorescent protein generated from the RFP of sea anemone Entacmaea quadricolor. TagRFP exhibits fluorescence with excitation/ emission maxima at 555/584 nm respectively, and brightness that is nearly three times higher than mCherry5.6.