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. Author manuscript; available in PMC: 2019 Oct 15.
Published in final edited form as: J Comp Neurol. 2018 Oct 15;526(15):2493–2508. doi: 10.1002/cne.24508

Figure 1: Cas9/CRISPR-mediated non-homologous end-joining insertion of donor plasmids upstream of predicted slc6a2 5’UTR generates NA-reporter lines.

Figure 1:

Donor plasmids (a) containing the MBait target sequence, a minimal heat shock promoter, and a membrane-targeted fluorescent reporter were coinjected with Cas9, an MBait sgRNA, and an sgRNA targeted approximately 300 bp upstream of the 5’UTR for slc6a2 (NET). Sequencing (b) revealed insertion near the target location with (TdTomato) and without (ECFP) small insertions. Deletions were present in both cases. Robust expression of ECFP (c; 4 dpf) and TdTomato (d,e; 6 dpf) was seen throughout the fish. Cell bodies were visible in the medulla oblongata, area postrema (d, yellow arrows) and LC (e, yellow arrows). Immunostaining (f-j) showed strong overlap between TH and reporter cells in the LC (f, orange boxes; g) and in the area postrema/medulla oblongata (h). TH but not reporter was seen in the dopaminergic diencephalon, as expected (j). Regions of reporter expression without TH expression were large blood vessels (e,f, green arrows), weakly expressing cells proximal to the LC (g, orange arrows) and an unidentified group of cells near the dorsal surface of the brain (i).