Supplementary Figure 2: The original GFP-dependent Cre recombinase (CRE-DOGOG).
From: Cell type–specific manipulation with GFP-dependent Cre recombinase
(a) CRE-DOGOG is made of N-CreintG and C-CreintG. CAG-loxP-Neo-loxP-luc2 (CALNL-luc2) is a Cre-dependent luciferase reporter. N-VMA and C-VMA are N- and C-terminal split intein portions from yeast VMA1 gene, respectively. GBP, GFP-binding protein. NLS, nuclear localization signal. (b-c) Luciferase assays showing specificity of CRE-DOGOG activity for each component of the system (b) and for different fluorescent proteins (c). n=9 replicate samples each condition (see Online Methods for n definition). In box plots, boxes show 25th to 75th percentile, line in box shows median, whiskers show minimum to maximum range. Scatter plots show median, interquartile range. (d-j) GFP directly controls CRE-DOGOG activity in a cell-specific manner in vivo. (d) Schematic of experiment. CAG-LNL-DsRed is a Cre-dependent reporter. CAG-nlacZ, which expresses n-βgal, is a substitution plasmid for GFP. (e-g) CRE-DOGOG induces expression of CALNL-DsRed reporter in a GFP-dependent manner in electroporated retinas. DsRed expression pattern depends on whether the broadly active CAG (e) or rod-specific Rho (f) promoters drives GFP expression. Scale bar, 20 μm. (h) Efficiency of CRE-DOGOG recombination in electroporated retinas. An electroporated cell is defined as either GFP+ or n-βgal+. Rod photoreceptors in the outer nuclear layer (ONL) were quantified. Sample size: GFP+ condition - At least 50 GFP+ cells/retina (n=3 retinas). Rho-GFP condition - At least 50 GFP+ cells/retina (n=4 retinas). No GFP condition - At least 100 n-βgal+ cells/retina (n=3 retinas). (i) GFP specificity of floxed DsRed+ ONL cells. Sample size: At least 30 to 50 cells/retina (CAG-GFP, n=3 retinas. Rho-GFP, n=4 retinas) (j) Expression pattern of floxed DsRed+ cells compared to GFP+ cells in ONL versus inner nuclear layer (INL). Sample size: CAG-GFP, at least 550 GFP+ cells/retina, at least 180 DsRed+ cells/retina, n=3 retinas; Rho-GFP, at least 200 GFP+ cells/retina, at least 100 DsRed+ cells/retina, n=4 retinas. For (e-j), consistent results were obtained in two independent experiments.
