Immunocytochemical analysis of the split-CreERT2 protein combination NCre-ERT2 + ERT2-CCre (NE+EC).

<p>A, B: CHO cells cultured in 24-well cell culture plates were transfected with NE+EC (150 ng of each plasmid/well) along with a reporter plasmid which expresses EGFP only after Cre-mediated DNA recombination (400 ng/well) and an expression vector coding for nuclear DsRed (100 ng/well; shown in red). Cells were cultured in the absence (A) or presence of 4OHT (1 µM, B). Recombination is detected by EGFP reporter expression (green; visualized by immunostaining using anti-GFP-antibodies). In blue, DAPI-staining of all nuclei is shown. C–F: Immunocytochemical confirmation of expression of NE and EC and of recombination in NE+EC transfected cells after application of 4OHT. CHO cells were transfected with NE+EC (400 ng of each plasmid/well) along with a reporter plasmid which expresses EGFP only after Cre-mediated DNA recombination (400 ng/well) and cultured in the presence of 4OHT (1 µM). NE (C), EC (D) and EGFP (E) were visualized by immunostaining for Flag-tag, Myc-tag or GFP, respectively, and detected by Cy5-, Cy3- or Cy2-conjugated secondary antibodies. F shows the merged images with NE in magenta, EC in red, EGFP in green and DAPI in blue. Note the additional nuclei stained with DAPI but not by anti-Flag- or anti-Myc-antibodies, which are also negative for EGFP. The bar in F corresponds to 20 µm and applies to all panels.</p

Split-CreERT2 mediated DNA recombination analyzed in CHO cells by luciferase assays.

<p>A: CHO cells cultured in 96-well cell culture plates were transfected with the different combinations of split-CreERT2 plasmids (for abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone-0008354-g001" target="_blank">Fig. 1</a>; 100 ng of each plasmid/well) and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Cre-dependent DNA recombination was analyzed by cotransfection of a CMV-LoxP-STOP-LoxP-luciferase reporter plasmid. Luciferase activity was normalized to luciferase activity in N+C transfected cells cultured in the absence of 4OHT. B: Induction ratios were calculated as [(luciferase activity in the presence of 4OHT)/(luciferase activity in the absence of 4OHT)]. The dotted line denotes a ratio of 1, which means no induction. C: A functional index of each combination of split-CreERT2 proteins was calculated as [(induction ratio)×(luciferase activity in the presence of 4OHT)]. This functional index is highest if the combination of split-CreERT2 proteins shows high inducibility and high activity in the presence of 4OHT. The figure shows data from 3 independent experiments, each of which was performed at least in triplicate. n.c.: not calculated.</p

Strategy for the generation of GAD65-tdTomato mice expressing the fluorescent protein tdTomato under control of the GAD65 promoter in GABAergic neurons.

<p><b>A:</b> Schematic representation of the BAC clone RPCI 23-407K8 (208,325 bp in total) containing the mouse full-length gene GAD65. Locations of PCR products for BAC verification (5’A-C; 3’A-B; Ex1) are indicated. <b>B:</b> Structure of the wild type GAD65 gene as well as the targeting construct. The endogenous start codon is located within exon 1. PCR reaction with primers (Ex1) flanking exon 1 results in a DNA fragment of 1012 bp in the wild type gene. The transgene consists of the ORF of tdTomato, a SV40-PolyA site as well as a FRT-flanked neomycin resistance cassette (neo) and was inserted directly after the endogenous ATG using homologous recombination provoked by homology arms (HA) indicated. <b>C:</b> Representation of the modified BAC containing the GAD65-tdTomato transgene after removal of the neomycin resistance cassette using Flp recombination. PCR using the same primers (Ex1) flanking exon 1 results in a product of 2746 bp in the modified BAC. <b>D:</b> PCR verification of the identity and integrity of the BAC using primers located 5‘ and 3‘ of the GAD65 gene (5’A-C; 3’A-B) as well as verification of the targeted modification site using primers spanning exon1 (Ex1).</p

Detailed characterization of the split-CreERT2 protein combination NCre-ERT2 + ERT2-CCre (NE+EC).

<p>A: CHO cells were transfected with different amounts of NE+EC and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Recombination was analyzed by luciferase reporter expression. The luminescence of reporter-only transfected cells (1.8±0.3%; n = 15) was subtracted from all values. B: CHO cells were transfected with 100 ng each of NE+EC and cultured in the presence of different concentrations of 4OHT. Luminescence in A and B was normalized to the luminescence observed in N+C transfected cells (100 ng of each plasmid/well) in the absence of 4OHT, which was set as 100%. C: PC12 20.4 cells were transfected with different amounts of NE+EC and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Recombination was analyzed by EGFP reporter expression using flow cytometry. The percentage of EGFP⁺-cells observed in the absence of NE+EC (4.4±0.7%; n = 9) was subtracted from all values. D: Cells were transfected with NE+EC plasmids (400 ng each/well) and cultured in the presence of different concentrations of 4OHT. The number of EGFP-positive cells in C and D was normalized to the number of EGFP-positive cells in N+C transfected cultures (400 ng of each plasmid/well) in the absence of 4OHT, which was set as 100%. All panels summarize data from a minimum of 3 independent experiments, each of which was performed at least in triplicate.</p

Identification of GABAergic and glycinergic neurons in TgN(GAD65-tdTomato) x TgN(GlyT2-EGFP) double transgenic mice by several microscopic techniques.

<p><b>A:</b> Overview of a frontal brain slice of a double transgenic 30 day old mouse showing tdTomato (red) expressing GABAergic and EGFP (green) expressing glycinergic neurons. The image was acquired using epifluorescence. Scale bar: 1 mm. <b>B:</b> High magnification epifluorescence image. <b>C:</b> GABAergic and glycinergic cells expressing tdTomato or EGFP, respectively, can also be visualized using confocal imaging. <b>D:</b> Also by using 2-photon laser scanning microscopy, tdTomato- and EGFP-fluorescence can be observed allowing for unequivocal identification of GABAergic and glycinergic neurons, respectively. Scale bar in D corresponds to 40 μm and applies to B-D.</p

Western Blot analysis of the expression of the different split-CreERT2 constructs.

<p>CHO cells were transfected with the different combinations of split-CreERT2 plasmids as indicated. Expression of N, NE and EN was analyzed by immunoblotting with anti-Flag antibodies, whereas C, CE and EC were detected by anti-Myc as well as anti-Cre antibodies. GAPDH was used as a loading control. The observed sizes of the proteins were as expected (N: 14.8 kDa; NE: 50.1 kDa; EN: 51.1 kDa; C: 41.6 kDa; CE: 77.0 kDa; EC: 78.0 kDa; GAPDH: 35.9 kDa).</p

Expression of tdTomato in Purkinje cells in the cerebellum.

<p><b>A:</b> Overview of the cerebellum of a 2.5 month old animal showing that only a subpopulation of Purkinje cells expressed tdTomato. <b>B, C:</b> In these Purkinje neurons both the dendritic tree as well as the axons (arrowheads) are clearly visible. Scale bars: 500 μm (A), 50 μm (B), 20 μm (C).</p

Overall expression pattern of tdTomato in GAD65-tdTomato transgenic mice at different developmental stages.

<p>In sagittal brain slices, a high reporter expression was observed in the olfactory bulb and striatum as well as in cortex, hippocampus, brainstem and substantia nigra in all investigated developmental stages (A: P1, B: P3, C: P10, D: adult 2.5 months). Fluorescence intensity of cells varied highly within one brain region and between different areas of the brain as well as different developmental stages. Scale bars: 500 μm.</p

Split-CreERT2 constructs.

<p>The original, non-inducible split-Cre proteins NCre and CCre have recently been described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone.0008354-Hirrlinger1" target="_blank">[15]</a>. To generate NCre-ERT2 and CCre-ERT2, the ERT2-domain was fused to the C-terminus of NCre and CCre, respectively. Vice versa, the ERT2-domain was fused to the N-terminus of NCre and CCre to obtain ERT2-NCre and ERT2-CCre, respectively. The abbreviations for the different constructs used in this paper are given in brackets. The thick vertical line at the N-terminus of all constructs denotes the position of the corresponding immunotag (NCre containing constructs: Flag-tag; CCre containing constructs: Myc-tag).</p

Identification of parvalbumin-, calretinin- and somatostatin-expressing subpopulations of interneurons among tdTomato-expressing neurons in transgenic mice.

<p>Immunohistochemical analysis showed the colocalization of the interneuron markers parvalbumin (A, B), calretinin (C, D) or somatostatin (E, F) with tdTomato fluorescence (red) in the cortex (A, C, E) and hippocampus (B, D, F) of 2.5 month old mice. Right panels show the overlay of tdTomato (red), parvalbumin, calretinin or somatostatin (green) as well as nuclei stained with DAPI (blue). Arrows highlight examples of cells expressing both tdTomato and either parvalbumin, calretinin or somatostatin. The scale bar corresponds to 50 μm and applies to all panels. <b>G:</b> Quantification of the relative contribution of parvalbumin-, calretinin- and somatostatin-expressing neurons to the number of cells expressing tdTomato.</p