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. 2017 Oct 4;4(5):ENEURO.0181-17.2017. doi: 10.1523/ENEURO.0181-17.2017

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Copyright © 2017 Tomorsky et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 1. — Upper cortical layer-enriched neuronal expression of HA-UPRT. A, Diagram of the CA>GFPstop>HA-UPRT transgene (Gay et al., 2014). B, Expression of the CA>GFPstop>HA-UPRT transgene (Gay et al., 2013) crossed to a Sepw1-cre line in neurons and endothelial cells (40× objective, scale bar: 10 μm). Green arrow: GFP-positive, Cre-negative neuron; white arrow: GFP-positive, Cre-negative endothelial cell; red arrow: UPRT:HA-positive, Cre-positive neuron. C, Sepw1-cre drives UPRT expression in layer 2/3 and to a lesser extent layer 4. Immunostaining for HA at P12 in a Sepw1-cre; CA>GFPstop>HA-UPRT cross reveals layer 2/3-enriched expression (10× objective, scale bar: 500 μm). DAPI is included to show cortical structure. D, Nr5a1-cre drives expression in a sparse subset of layer 4 neurons. Nr5a1-cre crossed to a tdTomato marker at P12 is specific to layer 4 (10 objective, scale bar: 500 μm). DAPI is included to show cortical structure. E, F, UPRT: HA is expressed in visual cortex of Sepw1-cre (E) or Nr5a1-cre (F) mouse lines crossed to the CA>GFPstop>HA-UPRT line (5× objective, scale bar: 500 μm). G, H, Sepw1-cre and Nr5a1-cre drive expression in neurons found predominantly in layer 2/3 or layer 4 of visual cortex, respectively. Neurons immunostained for the HA epitope on UPRT (red) in Sepw1-cre; CA>GFPstop>HA-UPRT or Nr5a1-cre; CA>GFPstop>HA-UPRT crosses, express the upper layer neuronal marker Cux1 (blue) (40X objective, scale bar: 50 μm). I, TU-tagging workflow using poly-A priming for cDNA synthesis and Nextera XT for library preparation.