Figure 4: Application of CGRP as a genetically expressed vasoactive reporter.

(a) Lentiviral vectors were used for expression of prepro-CGRP or a control construct. Top: probe vector controlled by the hEF1α promoter, encoding human prepro-CGRP (prepro-hCGRPα), an internal ribosome entry site (IRES), and the red fluorescent protein mKate2 joined by a self-cleaving viral 2A peptide to the blasticidin selection marker (Bla). Bottom: control vector lacking prepro-hCGRPα and the IRES sequence. (b) In vitro demonstration of substantial CGRP release from transfected HEK293FT cells. RAMP1/CLR activation by aliquots of purified CGRP peptide (light blue) or supernatants from cultured cells expressing control (grey) and prepro-CGRP (dark blue) lentiviral constructs was measured using the bioassay of Supplementary Fig. 2. Cell supernatants were assayed at 0 and 24 h after an initial washing, and all samples were assayed at tenfold dilution. Error bars denote s.d. of n=3 replicates. (c) In vivo detection of implanted HEK293FT cells producing CGRP in rat brains. A representative T₂-weighted MRI scan obtained 24 h after striatal injection of 3 × 10⁵ CGRP-producing or control cells (labels above) shows discernable signal enhancement consistent with probe-induced vasodilation in the CGRP injection region (dashed box) but not near the control injection site opposite. (d) Left: group-averaged map (left) displaying percent signal change (%SC, colour scale) observed between 0 and 24 h after cell implantation, averaged over five animals. Right: mean %SC in the square regions of interest superimposed on the image. Error bars denote s.e.m. across five animals. (e) Correspondence of MRI signal change (left, colour scale as in d) measured in vivo and mKate expression (right, red superimposed over a bright field image) visualized postmortem in a representative animal. The field of view corresponds to the rectangular box in c. Scale bar, 2 mm.