A new transgenic mouse model for functional tracing of circulation via albumin-tagged fluorescent probes

Liver-secreted albumin is the most abundant protein in blood plasma and cerebrospinal fluid. We have previously developed liver-targeting adeno-associated viral vectors (AAV8-P3-*) that express fluorescent protein-tagged albumin to visualize blood plasma in adult mice (DOI: 10.1016/j.crmeth.2022.100302). We have also established a virally induced CRISPR/Cas9-based knock-in of green fluorescent albumin in neonatal mice (DOI: 10.1101/2023.07.10.548084). Here, we have generated a new transgenic mouse model by CRISPR/Cas9 in which the bright red fluorescent protein mScarlet is knocked into the albumin locus to produce mScarlet-tagged albumin (Alb-mSc). In adult heterozygous knock-in mice, the plasma fluorescence signal intensity recorded an order of magnitude higher than a standard AAV-expressed Alb-mSc (AAV8-P3-Alb-mSc, 2E11 vg). This strong plasma fluorescence allowed for the imaging of the entire depth of the cortical vasculature reaching to the white matter (up to ~1mm in depth) by two-photon microscopy. Thin skull preparation enabled the visualization of dura mater by “shadow imaging”, where Alb-mSc presumably infiltrated into the interstitial space from dural vessels. Furthermore, Alb-mSc mice were crossed with Prox1-eGFP mice to delineate lymphatic structures. Alb-mSc signals in subdural interstitial space were clearly attenuated reflecting the composition of cerebrospinal fluid. Nonetheless, shadow imaging was possible in the cortical parenchyma, whereby cortical neurons are detected by the relatively high fluorescence of the extracellular space. The knock-in mouse line offers multi-purpose utilities for studying morphological and functional changes of vasculature in the brain and other organs.