• Constructed AAV-G22-Cre PRISM (adeno-associated viral) vectors and packaged in the neurotropic AAV-PhB.eB serotype with high titer and purity for non-invasive in vivo application (intravenous injection) in mice.
• We have determined the dose-response curve and the baseline dose of AAV-G22-Cre PRISM to report the spontaneous DNA damage response in the mouse brain. We tested the gene dosage dependency of the genetic sensor with G22 repeats. We examined 6 different doses (5x1011, 1x1011, 5x1010, 1x1010, 0.5x1010 VG/mouse) in vivo. We have determined the best titer/dosage of 1x1010 VG/mouse for in vivo application.
• We acquired Th-Cre, CamKII-Cre, D1-Cre, D2-Cre, Aai9 mouse breeding pairs from the Jackson Laboratory (JAX). We have been breeding these mice in C57BL/6J background. We already accumulated a sufficient number of mice for the simulated space radiation exposure at the NASA Space Radiation Laboratory (NSRL) this October 2021. • We successfully constructed the genetic sensor (biodosimetry) of neuronal genomic instability and validated the Cre dependency in vitro. These sensors will be used to determine space radiation-induced neuronal genomic instability in the most vulnerable neuronal cell types in multiple neurodegenerative disorders.
• We generated a quantitative ratiometric sensor: AAV-G34/G22/G13- mScarlet/fWasabi/fBFP PRISM vector and packaged it in AAV-PhB.eB serotype in high titer and purity for non-invasive in vivo application (intravenous injection) in mice.
• Intravenous systemic administration of AAV-G34/ G13- mScarlet/ fBFP PRISM sensor in different Cre driver lines (Drd1a. Drd2, TH Cre, CamkII Cre lines) was performed and we successfully labeled major neuronal cell types vulnerable to neurodegeneration, striatal medium spiny neurons, nigral dopamine neurons, hippocampal and cortical pyramidal neurons.
• Developed assays to quantify and confirm the mechanism of action of Dn to induce neuronal genomic instability using T7E1, Next Generation Sequencing (NGS), and capillary electrophoresis.
• We have made significant progress in optimizing the volume imaging of the iDISCO cleared mouse brains labeled with the genetic sensor of neuronal genomic instability.
• We have demonstrated that radiation mimic, Bleomycin, can increase AAV-G22-Cre dependent expression of AAV-G34/ G13- mScarlet/ fBFP PRISM sensor (systemic administration) genetic labelling in the mouse brains.
• We have scheduled and are ready for our first beamline exposures on 10/29-10/20/2021.
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