Fig. 1: The design of IBIST and the in vitro and in vivo validation.
From: An intein-split transactivator for intersectional neural imaging and optogenetic manipulation
a Scheme depicting construct design of intein-split tTA. b Diagram depicting the reconstitution of tTA by intein-based protein splicing. c Amino acid sequence of intein-fused tTA fragments. d, Examples of green fluorescence in HEK293T cells 72 h after transfections of reporter plasmid (TRE-HTG) and intein-split tTA plasmids with EF1α promoter (tTAN, tTAC, both or neither). e Examples showing the fluorescence of HTG reporter (shown in green) and tTAN (BFP tagged, shown in magenta) in HEK293T cells with all three plasmids transfected. Arrows: co-labeled cells. f Summary of green fluorescence intensity (arbitrary units, arb. units) of HEK293T cells (d) at 48 h and 72 h post-transfection (n = 4 field of view [FOV] in each group). One-way ANOVA: F(7,24) = 95.73, ****P = 5.67 × 10−16. Tukey’s multiple comparisons test: tTAC+tTAN group at 72 h is significantly higher than that at 48 h post transfection, ****P = 6.17 × 10−6; both are significantly higher than other groups at the same time post-transfection (****P < 0.0001 for all). g Percentage summary of cells labeled by HTG and tTAN in e (100 ± 0% co-labeled vs. 0 ± 0% reporter only; Mann-Whitney U test, *P = 0.029, n = 4 FOV). h Examples showing reporter fluorescence (AAV-TRE-HTG) after its injection into hippocampus and co-injections of AAVs (CaMKIIα promoter) for tTAN, tTAC, both or neither, respectively. i Examples showing fluorescence of tTAN (shown in magenta) and HTG reporter (shown in green) in hippocampal cells. Open arrows: reporter only. Filled arrows: co-labeled. j Summary of green fluorescence intensities in hippocampus (Each group, n = 6 FOV, N = 3 animals). One-way ANOVA between groups: F(3,20) = 20.57, P = 2.52 × 10−6. Turkey’s multiple comparisons: TRE-HTG + tTAC+tTAN group vs. others, ****P < 0.0001. k, Percentage summary of cells labeled by reporter and tTAN (88.4 ± 1.3% co-labeled vs. 11.6 ± 1.3% reporter only; paired t-test, ****P = 7.95 × 10−7, n = 6 FOV, N = 3 animals). Scale bars: 200 µm (h), 20 µm (d, i), 5 µm (e). Data summary: mean ± SEM. Statistical tests: two sided. Source data are provided as a Source Data file.
