In vivo direct cell-penetrating peptide mediated protein transduction system in Acyrthosiphon pisum.

OBJECTIVE: The principal delivery method for CRISPR-based genome editing in insects is now based on microinjection into single cells or embryos. The direct protein transduction systems cannot be employed in aphids because oogenesis occurs without apparent vitellogenesis. Given the limited timing of injection into the embryonic stage in oviparous aphids, a protein delivery system from the hemolymph to the germline and embryos would be a useful tool for genome editing. This study reports a newly developed direct protein delivery system for aphids using cell-penetrating peptides (CPPs). CPPs are short peptides that translocate across the plasma membrane when bound to cargo proteins. RESULTS: Penetratin (PEN), a widely conserved CPP among insects, was identified in this study. We used mVenus, a recombinant fluorescent protein, as a visual marker for CPP availability assessments, and fused it with PEN by bacterial protein expression. The mVenus-PEN recombinant proteins were introduced into the hemolymph of adult unwinged Acyrthosiphon pisum females using a nanoinjector. Fluorescence emitted by mVenus-PEN was observed in various tissues, such as the gut, trachea, bacteriocytes, and their progeny. This study shows that PEN can deliver exogenously expressed proteins into tissues in vivo, indicating that CPPs are powerful tools for protein transduction.

Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.