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. 2020 Apr 10;18(4):e3000665. doi: 10.1371/journal.pbio.3000665

Fig 8. Cre-dependent coexpression and labeling of two proteins in single neurons.

Fig 8

(A) Overview of plasmids used for Cre-dependent expression of mCherry-KASH or Synapsin-FLAG in knock-in neurons. (B) Examples of GFP-P2A knock-in–driven expression of mCherry-KASH or Synapsin-FLAG (Alexa568) (magenta) for various knock-ins. DIV 21. Scale bars, 10 μm and 5 μm for the overviews and zooms, respectively. (C) Overview of plasmids used for multiplex knock-in of two proteins in single neurons (ORANGE-CAKE). (D and E) Examples of β3-tubulin-GFP-P2A-Cre (green), Lox GluA1-HA (magenta, Alexa594) double knock-in, (D) and PSD95-GFP-P2A-Cre (green), Lox Halo-β-actin (magenta, JF549) double knock-in (E). Shown are overviews (confocal) and zooms (gSTED). DIV 21. Scale bars, 20 μm for the overviews and 5 μm (dendrites) and 500 nm (spine) for the zooms. (F) Examples of various combinations of GFP-P2A-Cre (green) and Lox (magenta) double knock-ins. HA was visualized by anti-HA staining (Alexa594), and Halo with Halo-JF549 ligand. DIV21. Scale bars, 10 μm and 5 μm for the overviews and zooms, respectively. Asterisk indicates enhancement with anti-GFP antibody (Alexa488). CAKE, conditional activation of knock-in expression; CaV, voltage-dependent Ca2+-channel; DIV, day in vitro; FLEx, flip-excision; GFP, green fluorescent protein; GluA, glutamate receptor AMPA; gRNA, guide RNA; gSTED, gated stimulated-emission depletion; HA, hemagglutinin; hSyn, human Synapsin; JF549, Janelia Fluor 549; KASH, Klarsicht, ANC-1, Syne Homology; ORANGE, Open Resource for the Application of Neuronal Genome Editing; PSD95, postsynaptic protein 95; T, target sequence.