Special Seminar: A Genetic Toolset for Investigating Glio-vascular Interactions

About this Seminar

Studying blood microcirculation is crucial for understanding vascular diseases. Blood flow is currently imaged following invasive administration of fluorescent dyes that disappear within one hour from injection. Dr. Hirase and his team took three new molecular genetic approaches for the longitudinal study of vasculature. Liver-secreted albumin is the most abundant protein in blood plasma and cerebrospinal fluid. They have developed liver-targeting adeno-associated viral vectors (AAVs) that express fluorescent protein-tagged albumin to visualize blood plasma in mice following a single systemic injection. While this approach is versatile for imaging in adult mice, the dilution of the episomal AAV genome in the rapidly growing liver in infants precludes chronic neonatal imaging. Therefore, they have established a virally induced CRISPR/Cas9-based knock-in of fluorescent albumin. They constructed an AAV that includes ~1 kb homologous arms around Alb exon 14 to express Alb-mNeonGreen instead of Alb. Systemic injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days and persistent expression of Alb-mNeonGreen for at least the next three months. As an alternative approach, they have used CRISPR/Cas9 genome editing to generate a mouse in which the bright red fluorescent protein mScarlet is knocked into Alb exon 14. This mouse line is also equipped with a signal amplification mechanism allowing for investigations in glio-vascular interactions. These three new plasma labeling approaches are expected to provide powerful means for micro- and macroscopic imaging of murine vasculature.

You May Also Be Interested In...