RT-PCR for ChR2 expression in DRG.

ChR2 expression in DRG neurons as measured by RT-PCR. β–Actin was used as a positive control to confirm successful protein extraction and equal loading of samples. All data are calculated as mean ± SD of 5 animals. *P V1.7iCre/+;Ai32/+ mice. †P = 0.007 and #P = 0.006, compared with NaV1.7iCre/iCre;Ai32/+ mice.</p

OPA test.

(a) Experimental schematic. (b) The OPA test was performed with the wild-type (WT) and mice of the four genotypes. The data were analyzed using one–way ANOVA followed by Bonferroni post-hoc analysis. All results are calculated as mean ± SD of 10 or more animals. *P < 0.001, compared with WT mice.</p

Fig 3 -

Paw withdrawal test (von Frey test) (a) and plantar test (b). (a) The von Frey test was performed with the wild type (WT) and mice of the four genotypes. The hind paw withdrawal data were analyzed using one-way ANOVA. All results are calculated as mean ± SD of 10 or more animals. Individual results for each strain are as follows: WT (B6J), 4.4 ± 0.7 g; NaV1.7iCre/+;Ai32/+, 4.8 ± 0.4 g; NaV1.7iCre/iCre;Ai32/+, 4.5 ± 0.8 g; NaV1.7iCre/+;Ai32/Ai32, 4.0 ± 1.2 g; and NaV1.7iCre/iCre;Ai32/Ai32, 4.3 ± 0.7 g. (b) The plantar test was performed with the WT and mice of the four genotypes. The data were analyzed using one-way ANOVA. All results are calculated as mean ± SD of 10 or more animals. Individual results for each strain were as follows: WT (B6J), 5.7 ± 0.8 s; NaV1.7iCre/+;Ai32/+, 5.5 ± 1.2 s; NaV1.7iCre/iCre;Ai32/+, 6.6 ± 1.7 s; NaV1.7iCre/+;Ai32/Ai32, 6.6 ± 2.2 s; and NaV1.7iCre/iCre;Ai32/Ai32, 6.3 ± 1.2 s.</p

Distribution of ChR2-EYFP channels in DRG.

The green fluorescent signal represents the direct fluorescence of ChR2-EYFP. (a) 20× and (b) 40×. (PPTX)</p

The original unadjusted and uncropped images of PCR presented in Fig 6A.

(PDF)</p

Fig 1 -

Schematic diagram of the Scn9aiCre knock-in allele (a) and PCR for genotyping founder mice (b). (a)The last exon of Scn9a is illustrated. The P2A-iCre sequence was inserted immediately after the stop codon of Scn9a. (b) The detection of the insert upstream of the 5′- and 3′-homology arm and the random integration of pX330-mC and piCre-Scn9a. Six founder mice were determined to carry the designed knock-in mutation. M, marker; P, positive control; N, negative control.</p

Light irradiation hind paw withdrawal test.

(a) Experimental schematic. (b) The blue light irradiation hind paw withdrawal test was performed in wild-type (WT) and mice of the four genotypes. The data were analyzed using one-way ANOVA followed by Bonferroni post-hoc analysis. All results are calculated as mean ± SD of 10 or more animals. *P †P = 0.03 and #P = 0.02, compared with WT mice.</p

Fig 2 -

Establishment of different lines of transgenic mice (a) and distribution of ChR2-EYFP channels in DRG (b). (a) NaV1.7iCre/+;Ai32/+ mice were created by crossing homozygous NaV1.7–iCre mice with heterozygous Ai32 mice. Subsequently, NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice were created by crossing NaV1.7iCre/+;Ai32/+ mice with each other. The four genotypes of mice used in the study are highlighted in yellow. (b) A typical immunohistochemical image showing of ChR2-EYFP in the DRG, the dorsal horn of spinal cord, and glabrous skin of NaV1.7iCre/+;Ai32/+ mouse was shown. Green and red fluorescence indicates ChR2-EYFP and NaV1.7, respectively. ChR2–EYFP were expressed on NaV1.7-expressing DRG neurons. Green fluorescence (ChR2–EYFP expression) can be observed in the dorsal horn. ChR2–EYFP is localized in free nerve endings in the lower and upper dermis of glabrous skin.</p

von Frey test and plantar test of Na_V1.7^iCre/iCre and Na_V1.7^iCre/+.

The von Frey test (a) and plantar test (b) were performed with wild-type (WT), NaV1.7iCre/iCre, and NaV1.7iCre/+ mice. The data were analyzed using one-way ANOVA. All results are calculated as mean ± SD of 10 or more animals. Individual results for each strain are (a) WT (B6J): 4.4 ± 0.7 g, NaV1.7iCre/iCre: 4.9 ± 0.8 g, and NaV1.7iCre/+: 4.9 ± 0.6 g, (b) WT (B6J): 5.7 ± 0.8 s, NaV1.7iCre/iCre: 5.5 ± 1.1 s, and NaV1.7iCre/+: 6.0 ± 1.8 s. (PPTX)</p