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bioRxiv · 10.1101/2023.07.16.549202

A novel polycistronic method tailored for engineering split GECIs

Olszakier, S.·Hussein, W.·Heinrich, R.·Andreyanov, M.·Otor, Y.·Schiller, J.·Kellner, S.·Berlin, S.
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Abstract

We 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 ORFs in 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 denote SPLIT--Stop-codon mediated Polycistronic Induction in HeTerologous expression systems-- for rapid and easy development of fragmented proteins by the sole use of stop codons. We validated the SPLIT method 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 excitation in vivo in 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 types in vitro and in vivo. We harness translation re-initiation to express multiple ORFs, to engineer rainbow indicators and to swiftly produce functional split-proteins and probes.

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Olszakier, S., Hussein, W., Heinrich, R., Andreyanov, M., Otor, Y., Schiller, J., Kellner, S., Berlin, S.. 2023-07-16. A novel polycistronic method tailored for engineering split GECIs. https://doi.org/10.1101/2023.07.16.549202

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