ai65 targeting vector  (Addgene inc)

Journal: Frontiers in Molecular Neuroscience

Article Title: A Na V 1.8 FlpO mouse enabling selective intersectional targeting of low threshold C fiber mechanoreceptors and nociceptors

doi: 10.3389/fnmol.2025.1574219

Figure Lengend Snippet: Verification of the Th CreERT2 ; Na V 1.8 FlpO ; Ai65 mouse line. (A) Strategy for Cre/Flp-dependent intersectional targeting. Crossing of Ai65 mice with Na V 1.8 FlpO and Th CreERT2 mice removes FRT-stop-FRT and (after tamoxifen administration) lox-stop-lox cassettes in cells co-expressing FlpO and CreERT2, enabling transcription of tdTomato in those cells. (B) Section of a lumbar DRG subjected to immunolabeling of tdTomato and TH, and to in situ hybridization for Scn10a . Arrowheads indicate cells triple positive for tdTomato, TH and Scn10a . Arrow indicates an example of a TH + / Scn10a + cell that lacks tdTomato expression. Scale bar, 50 μm. Micrographs are single optical sections obtained using a 20x/0.8 objective and 27 μm pinhole. (C) Quantification of recombination efficiency with respect to TH + / Scn10a + cells. Note that all TH + cells were Scn10a + and vice versa. Each data point corresponds to a single DRG section and mouse; n = 3 mice. Error bars indicate S.E.M.

Article Snippet: For intersectional genetic targeting of C-LTMRs, Na V 1.8 FlpO mice were crossed with either Ai65 mice (RCFL-tdT; Jackson Laboratory, strain #021875) ( ) or ROSA26DR-Matrix-dAPEX2 (Jackson Laboratory, strain #032764; here called APEX2 mice) ( ); the resulting offspring was further crossed with Th CreERT2 mice (Jackson Laboratory, strain #025614) ( ) to generate offspring heterozygous for Na V 1.8 FlpO , Th CreERT2 , and reporter.

Techniques: Expressing, Immunolabeling, In Situ Hybridization

Journal: Frontiers in Molecular Neuroscience

Article Title: A Na V 1.8 FlpO mouse enabling selective intersectional targeting of low threshold C fiber mechanoreceptors and nociceptors

doi: 10.3389/fnmol.2025.1574219

Figure Lengend Snippet: Targeting of C-LTMRs in spinal cord and skin of Th CreERT2 ; Na V 1.8 FlpO ; Ai65 mice. (A) Distribution of tdTomato + processes in the superficial dorsal horn of L4 spinal cord with respect to IB 4 binding and PKCγ and VGluT3 immunolabeling. Dashed line indicates ventral border of the IB 4 plexus. Note that the band of tdTomato + fibers and endings were almost entirely ventral to this border, and localized to the dorsal portion of the region inhabited by PKCγ neurons. Note that the medial gray matter receiving input from glabrous skin was devoid of tdTomato + fibers. tdTomato + processes showed near-complete co-localization with VGluT3 in lamina II. Scale bars are 100 μm except in bottom-rightmost panel, which is 20 μm. Single optical sections obtained using a 20x/0.8 objective and 24 μm pinhole except bottom-rightmost panel, which is a single optical section acquired using a 63x/1.4 objective with 28 μm pinhole. (B) Comparison of spinal distribution of tdTomato + processes in Th CreERT2 ; Na V 1.8 FlpO ; Ai65 versus Th CreERT2 ; Ai14 mice. In the former, tdTomato + processes were only observed in the superficial dorsal horn as well as in the dorsal root and Lissauer’s tract, whereas in Th CreERT2 ; Ai14 mice tdTomato + processes were present throughout gray and white matter. Insets show magnified false-color views of the regions in the deep dorsal horn indicated by dashed frames. Scale bar, 250 μm. Large views are widefield micrographs obtained using a 20x/0.5 objective; insets are single optical sections obtained using a 40x/1.3 objective and 36 μm pinhole. (C) Hairy skin innervation by tdTomato + fibers in Th CreERT2 ; Na V 1.8 FlpO ; Ai65 versus Th CreERT2 ; Ai14 mice. tdTomato + lanceolate nerve endings presumably formed by C-LTMRs were found around hair follicles in both mice (indicated by arrowheads). In Th CreERT2 ; Ai14 mice presumed sympathetic nerve fibers were also tdTomato + (arrows). Images are maximum intensity projections of 26 (upper panel) or 28 (lower panel) optical sections at 1.0 μm separation acquired using a 20x/0.8 objective and 30 μm pinhole. Scale bar, 50 μm.

Article Snippet: For intersectional genetic targeting of C-LTMRs, Na V 1.8 FlpO mice were crossed with either Ai65 mice (RCFL-tdT; Jackson Laboratory, strain #021875) ( ) or ROSA26DR-Matrix-dAPEX2 (Jackson Laboratory, strain #032764; here called APEX2 mice) ( ); the resulting offspring was further crossed with Th CreERT2 mice (Jackson Laboratory, strain #025614) ( ) to generate offspring heterozygous for Na V 1.8 FlpO , Th CreERT2 , and reporter.

Techniques: Binding Assay, Immunolabeling, Comparison

Journal: bioRxiv

Article Title: Mapping of dI3 neuron sensorimotor circuits across the cervical and lumbar spinal cord

doi: 10.1101/2024.11.17.624039

Figure Lengend Snippet: A. Diagram of genetic strategy for generating Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai80 (RCFL-CatCh)-D and Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai65 (RCFL-tdT)-D mice. Isl1 Cre +/- mice were crossed with Slc17a6 FlpO +/+ mice to drive the expression of Cre and FlpO recombinase in dI3 neurons and Vglut2 + nociceptive afferents, forming dI3-driver mice. For optogenetic experiments, Isl1 Cre +/- ; Slc17a6 FlpO +/+ (dI3-driver) mice were crossed with Ai80 (RCFL-CatCh)-D (CatCh) mice containing a frt -flanked STOP cassette and a loxP -flanked STOP cassette upstream of the CatCh/EYFP fluorescent protein, resulting in Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai80 (RCFL-CatCh)-D (dI3-driver-CatCh) mice. To visualize the dI3 neurons and Vglut2 + afferents, dI3-driver mice were crossed with Ai65 (RCFL-tdT)-D (TdTomato) mice containing a frt -flanked STOP cassette and a loxP -flanked STOP cassette upstream of the TdTomato fluorescent protein, resulting in Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai65 (RCFL-tdT)-D (dI3-driver-TdTomato) mice. B. Representative image of TdTomato (red) labelled dI3 neurons and Vglut2 + afferents in dI3-driver-TdTomato mice. To avoid activation of these afferents in dI3-driver-CatCh mice, a dorsal hemisection was performed at the indicated level. C. Diagram of the ex vivo spinal cord preparation. The spinal cord was isolated in neonatal P1-P7 dI3-driver-CatCh mice, and a dorsal hemisection was performed at the cervical (C1-C8) and lumbar (L1-L5) segments to remove Vglut2-CatCh + afferents. Three suction electrodes were simultaneously applied to various ventral roots to record from C4-C8 and L1-L5 segments, and blue light was applied to ipsilateral segments. Within the same animals, blue light was also independently applied to contralateral segments from C4-C8 and L1-L5.

Article Snippet: To visualize the dI3 neurons and Vglut2 + afferents, dI3-driver mice were crossed with Ai65 (RCFL-tdT)-D (TdTomato) mice (The Jackson Laboratory, Strain No.: 021875) containing a frt -flanked STOP cassette and a loxP -flanked STOP cassette upstream of the TdTomato fluorescent protein, resulting in Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai65 (RCFL-tdT)-D (dI3-driver-TdTomato) mice.

Techniques: Expressing, Activation Assay, Ex Vivo, Isolation