Periodic Reporting for period 2 - MAGNIFISCENT (mesoscale multi-mode MRI of molecular targets)
Período documentado: 2021-07-01 hasta 2022-12-31
Imaging defined cells, over extended time, depends on signal strength, stability, accessibility and specificity. Whereas light-microscopy (LM) can provide these, it does not allow imaging of entire intact tissues; imaging-depths and area-size are restricted, and not easily obtained through skin and bone. Magnetic Resonance Imaging (MRI) outperforms LM in these instances, providing images of large-fields-of-view (i.e. mesoscale), at any depth, easily across bone. Nevertheless, MRI suffers from low signals, spatial resolution and cannot detect specific biological targets such as defined cellular populations.
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 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).
To meet our objectives, we have made substantial progress in two aspects. In the chemical part of our project, we have developed novel means for screening of novel contrast agents ex vivo by MRI and LM (manuscript in preparation). Briefly, to image entire tissues (without sectioning) by LM, opaque tissues such as the brain, need to undergo optical clearing (chemical clearing rendering the brain transparent). However, thus far, cleared-brains could not be jointly imaged by MRI (they lose all contrast). We explored MRI compatibility of multiple clearing techniques and were able to develop a process that renders two different clearing techniques compatible for ex vivo MR-Imaging. In the process, we were also able to determine the major source for contrast in ex vivo MRI, a highly debated and elusive feature.
A novel polycistronic method tailored for engineering split proteins
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.
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.