Periodic Reporting for period 2 - MAGNIFISCENT (mesoscale multi-mode MRI of molecular targets)
Période du rapport: 2021-07-01 au 2022-12-31
We are developing an innovative approach to perform multimodal imaging in vivo and ex vivo. Though applicable to any tissue, we focus on the brain. Our patented approach is intended to provide users with the ability to study emergence of diseases, with high resolution and target validity; empowering early detection of diseases that affect cellular populations, such as neurodegenerative diseases—notably Alzheimer’s disease. This should have a significant impact on the study and detection of many types of diseases, especially of the brain.
Our key goals are:
• Genetic-MRI— To image defined targets by MRI at great resolution
• multicolor-MRI— To detect several defined cellular targets jointly by MRI.
• MRI-interactome— To image cellular-interactions by MRI.
• ENTRAP-MRI— To image committed neurons destined for degeneration (apoptosis).
We assessed the feasibility of using stop-codons as means to obtain polycistronic expression in eukaryotic cells. We show robust 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, which guided us to develop a technicolor Genetically-Encoded Functional Rainbow Indicators; GEFRIs for monitoring cellular morphology, neuronal firing and cellular stress, concomitantly. These findings further guided us to develop a new technique we denote SPLIT—Stop-codon mediated Polycistronic Induction in Terologous expression systems— for rapid and easy development of fragmented proteins by the simple introduction of stop codons within ORFs. We first validate the SPLIT method by generating several new split-GFP variants, then engineer a palette of functional split-GCaMP6 variants and, lastly, generate a split ca2+-probe localized at ER and mitochondria junctions. Together, we explore non-canonical translation mechanisms and show these to be highly prevalent in various cell types. We harness translation re-initiation to express multiple ORFs, to engineer rainbow indicators and to swiftly produce functional split-proteins and probes.