Fig 1.
Strategy for CRISPR-directed knockin targeting Rosa26 Tac1 and Plekhg1 loci.
(a) Schematic representation of the Rosa26 locus showing the positions of the normal XbaI targeting site and the homology arms; below, the sequence around the XbaI site highlights the sgRNA used for CRISPR targeting. (b) The strategy used for CRISPR-mediated homologous recombination is highlighted for Rosa26 targeting constructs. (c) The two Rosa26 targeting constructs used a standard vector that was modified by deletion of the sgRNA recognition sequence, addition of a directional SfiI cloning site between the loxP-flanked transcriptional stop site and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and deletion of selection markers. Schematic representations of the strategy used for CRISPR directed generation of (d) the Tac1-tagRFP-2A-Tva and (e) Plekhg1-2a-FlpO mice; the genes indicating homology regions used (black boxes) and the sequence of the sgRNA (italic, bold) and PAM-site (green) used to direct CRISPR cleavage shown above a diagram of the construct used for homologous recombination highlighting the fusion junctions between homology arms (black) and knockin-genes (red). Relevant starting ATGs (Tac1) and stop codon TAG (Plekhg1) are highlighted in blue.
Fig 2.
Characterization of Rosa26-CAG-lox-stop-lox-TVA mice.
(a) Strategy and (b, c) results of testing heterozygous Rosa26-CAG-lox-Stop-lox-TVA mice for Cre-dependent expression of TVA. (b, c) Confocal images (combined red and green channels) of sections through the region of the brain injected with (b) AAV-hSyn-Cre or (c) AAV-hSyn-eGFP followed in each case by EnvA G-depleted Rabies-mCherry demonstrate that after Cre-recombination (b) EnvA-pseudotyped Rabies infected neurons in the forebrain but, (c) without Cre-expression no infection was observed; scale-bar 100 μm.
Fig 3.
Characterization of Rosa26-CAG-lox-stop-lox-extracellular-Ruby2-sm-ollas mice.
(a) Schematic representation of the synthetic construct used for cell surface expression of the ollas antigen: the mouse Tas1R3 signal peptide was attached to a non-fluorescent mRuby-ollas spaghetti monster that was fused to the CD4 transmembrane domain. (b) Confocal images of tissue sections and cells immune-stained using anti-ollas immunofluorescence (red); tissue sections were counterstained with DAPI (blue). Note that as expected, no expression of the ollas-tagged mRuby was detected in the trigeminal ganglion of Rosa-CAG-lox-stop-lox-extracellular-ollas-tagged mice in the absence of a Cre-transgene (upper-left panel) or in the brain of the knockin crossed into a Trpv1-Cre background (upper-right panel). In contrast, the trigeminal ganglia of these double positive mice (lower-left panel) prominently express mRuby-ollas; note that many small and medium diameter cells express the ollas-tag, scale-bar = 50 μm. Lower-right panel, trigeminal ganglion cells were isolated by enzymatic digestion and were stained without permeabilization; positive cells (red) can be distinguished from debris and non-labelled cells in the superimposed transmitted light image (gray), scale bar = 50 μm.
Fig 4.
Characterization of Tac1-tagRFP-2A-Tva mice.
Fluorescence images of sections of sensory ganglia and regions of the brain illustrate the cellular expression of tagRFP (red). (a) Representative section of the trigeminal ganglion, immunostained for Tac1 (green) and counter-stained with DAPI (blue) demonstrates that tagRFP (red) is expressed in the subset of trigeminal neurons that stain positive for Tac1. In (b) the geniculate ganglion, (c) the brainstem (nucleus of the solitary tract), (d) the parabrachial nucleus and (e) the para-subthalamic nucleus (PSTN), tagRFP (red) is also expressed in subsets of neurons. Note the strongly positive fiber tracts (arrowed) running close to the PSTN; scale bar = 100 μm.
Fig 5.
Characterization of Plekhg1-2A-FlpO mice.
(a) Section through the thalamus of a Plekhg1-2A-FlpO mouse stained by in situ hybridization with probes for Plekhg1 (green) and FlpO (red); nuclei are counterstained with DAPI (blue). Note that Plekhg1 (and FlpO) expression is limited to a subset of cells in the thalamus; neighboring brain regions (e.g. below and to the right of the dotted line) do not express these transcripts. (b-d) Strategy characterizing Plekhg1-directed expression of FlpO-activity. Heterozygous mice were injected with a Flp dependent reporter virus as indicated schematically in (b). (c, d) Fluorescence images of a section through the thalamus of an injected mouse illustrates the injection tract (red) and FlpO dependent expression of eYFP (green) in (c) a stitched image of the entire section and (d) a magnified view of the boxed region to highlight cellular expression; scale bars (a) = 50 μm (c) = 500 μm, (d) = 100 μm.