Fig. 6
Retrofitting a Transgenic GFP Mouse Line for GFP-Dependent Manipulation of Gene Expression and Neural Circuit Activities (A) Tg(GUS8.4GFP) expresses GFP in type 7 cone bipolar and rod bipolar cell types (green fill) of the retina. Adopted schematic (Ghosh et al., 2004). (B) Cryosection of electroporated Tg(GUS8.4GFP) retina expressing Gal4-GBP2p65-GBP7 and UAS-tdT. Scale bar, 20 μm. (C) Type 7 (left) and rod bipolar (right) cell types labeled by UAS-tdT. Anti-Calretinin (left) or anti-Calbindin (right) staining identify specific layers of the IPL. Scale bar, 10 µm. GFP was immunostained in (B and C). (D) Schematic of ChR2 experiment. Electroporated Tg(GUS8.4-GFP) retinas expressing 10×UAS-ChR2/H134R-mCherry and 5×UAS-tdT were analyzed for ChR2-mediated responses in random GCL cells. (E) Cumulative plot of ON responses in GCL cells. Number of spikes counted during the first 300 ms after stimulus onset, normalized to control (minus APB). APB blocks ON responses originating from photoreceptors. Plots are mean ± SEM (n = 4 per condition). (F) Spiking response of a GCL cell. Gray bar, duration of light stimulus. Response to normal light stimuli under control condition (top) or in the presence of APB (middle). Light stimuli focused on INL activate ChR2/H134R in the presence of APB (lower). (G and H) Top and side views of a neurobiotin-filled (green) ganglion cell identified by light stimulation of ChR2. Magenta lines indicate level of anti-Chat bands (not shown). Scale bar, 20 μm. |
Reprinted from Cell, 154(4), Tang, J.C., Szikra, T., Kozorovitskiy, Y., Teixiera, M., Sabatini, B.L., Roska, B., and Cepko, C.L., A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation, 928-939, Copyright (2013) with permission from Elsevier. Full text @ Cell