Fig. 1: Development of the rAAV-miniBEND system for brainEC-specific labelling.
a, Schematic diagram illustrating the development of the rAAV-miniBEND system. MSA, multiple sequence alignment. Conserved regions, yellow. b,e, Schematic diagram depicting the truncation strategy for the Tek promoter (b, truncations for mouse promoter mPro) and cis-element (e). UTR, untranslated region. TSS, transcription start site. c,f, Bar chart displaying the transduction efficiency mediated by various truncated versions of the promoter (c, with mCis700) and cis-element (f, with mPro973). Light-green bars, cortex; sky-blue bars, hippocampus. Labelled EC density indicates the number of labelled endothelial cells per mm2 per 1011 AAV particles, with multiple regions for cell counting and statistical analysis. n = 8–12 regions from 2–3 mice in c and f. d,g, Representative images of fluorescent protein expression (EGFP/tdTomato) mediated by various truncated versions of the promoter (d) and cis-element (g) contained in rAAVs shown in b and e. h, Representative images of EGFP expression and co-labelling with anti-GLUT1 and anti-CD31. rAAV-PHP.V1-mPro973-Cre-mCis1349 was administered to Ai47 via retro-orbital injection. Blue arrowheads, endothelial cells. i, Representative images of EGFP expression and co-labelling with antibodies against marker proteins of pericytes (Pdgfrβ and NG2) in brain sections from Ai47 mice administered AAV-PHP.eB-mPro973-Cre-mCis1349 via retro-orbital injection. Blue arrowheads, pericytes. j, Representative images of EGFP expression and labelling with antibodies against αSMA (arrowheads, blue), in brain sections from Ai47 or NG2DsRedBAC::Ai47 mice injected with AAV-PHP.eB-miniBEND-Cre. Red, the DsRed signal. k, Representative images showing the transduction pattern with rAAV using AAV-PHP.eB serotype virus with different recombinant vectors in the cerebellum: CAG-mScarlet, mPro973-Cre, mPro760-Cre and mPro723-Cre. The Tek cis-regulatory element used in the latter three groups was mCis700. These viruses were injected into wild-type mice or Ai transgenic reporter lines Ai47 or Ai14 as indicated. Blue arrowheads, neurons. l,m, The non-specific labelling ratio (l) and EC labelling efficiency (m) evaluated based on the labelling density in each transduced mouse (n = 10 regions per group). n = 8–12 regions from 2–3 mice in c, f and l, and unpaired two-sided Welch’s t-test (m). Data indicate the mean ± s.e.m.; ****P < 0.0001; ***P < 0.001. Panel a created with BioRender.com.
