Abstract
AbstractWe assessed the feasibility of using stop-codons as means to obtain polycistronic expression in eukaryotic cells. We show robust bicistronic expression of different open reading frames (ORFs), when these are cloned in-sequence and simply separated by stop codons (in-or out-of-frame), in heterologous expression systems and primary neurons. We further find this method to support polycistronic expression of three stop-codon-separated ORFsin vivo, which guided us to develop a technicolor Genetically-Encoded Functional Rainbow Indicators (GEFRIs) for monitoring cellular morphology and neuronal firing, concomitantly. These findings guided us to develop a new technique we denoteSPLIT—Stop-codon mediatedPolycistronicInduction in HeTerologous expression systems— for rapid and easy development of fragmented proteins by the sole use of stop codons. We validated theSPLITmethod by generating several new split-GFP variants, then engineer a palette of functional split-GCaMP6s variants and, lastly, generate a split ca2+-probe localized at ER and mitochondria junctions, denoted split-MEGIC. With the use of the probe, we show presence and activity of mito-ER contact sites within individual dendritic spines. Split-MEGIC can thereby be imaged by two-photon excitationin vivoin mice brains and, by standard confocal microscope in transgenic zebrafish larvae. Together, we explore non-canonical translation mechanisms and show these to be highly pervasive in various cell typesin vitroandin vivo. We harness translation re-initiation to express multiple ORFs, to engineer rainbow indicators and to swiftly produce functional split-proteins and probes.
Publisher
Cold Spring Harbor Laboratory