Fig 1.
Development of Gnao1+/G203R mouse model.
(A) Targeting of the Gnao1 locus. The location of the gRNA target protospacer and the PAM, and double stranded breaks following Cas9 cleavage are indicated on the WT allele. Deleted or modified sequences are highlighted in blue. The resulting edited allele sequence and translation are presented along with the sequences used as references for ssODN synthesis. (B) Heterozygous Gnao1+/G203R mutant mice are largely normal in size and behavior. Photo comparing mutant mouse with its littermate control is shown. (C) Gnao1+/G203R mice have a relatively normal survival; while homozygous Gnao1G203R/G203R mice die perinatally (P0-P1). (D) Gnao1+/G203R mice develop normally and gain weight similarly to their WT littermate controls.
Table 1.
Location, sequence and genotyping of gRNA targets in Gnao1 locus.
Fig 2.
The timeline for utilizing animals in this study.
Open field, Rotarod and Grip strength tests were performed on the same group of 8-week-old animals in this as showed above. DigiGait tests were done on naïve 8-week-old animals. After completion of the motor behavior studies, animals were used for the PTZ kindling study.
Fig 3.
Female Gnao1+/G184S mice and male Gnao1+/G203R mice show reduced time on RotaRod and reduced grip strength.
(A&B) Quantification of RotaRod studies. (A) Female Gnao1+/G184S mice lose the ability to stay on a RotaRod (unpaired t-test; ***p<0.001), while male Gnao1+/G184S mice appeared unaffected. (B) Male Gnao1+/G203R also showed reduced motor coordination on RotaRod (unpaired t-test, *p<0.01). (C&D) Quantification of grip strength results. Scores for each mouse were normalized to body weight. (C) Female Gnao1+/G184S mice are less capable of lifting weights compared to their Gnao1+/+ siblings (unpaired t-test, *p<0.05). (D) Both male and female Gnao1+/G203R mice showed reduced ability to hold weights (unpaired t-test, *p<0.05). Data are shown as mean ± SEM.
Fig 4.
G184S mutant mice showed reduced activities in open field test but G203R mutants don’t.
(A&C) Female and male Gnao1+/G184S mice showed decreased activity in the open field test. A total of 30 min activity was recorded which was divided into a Novelty (0–10 min) and a Sustained (10–30 min) period. (A) Representative heat map of overall activity comparing Gnao1+/+ and Gnao1+/G184S mice of both sexes. (C) Quantitatively, both male and female Gnao1+/G184S travelled less in the open field arena (2-way ANOVA; ****p< 0.0001, **p<0.01, *p<0.05). (B & D) Neither male nor female Gnao1+/G203R mice showed abnormalities in the open field arena. (B) Sample heat map tracing of female and male mouse movement in open field. (D) Quantification showed no difference between Gnao1+/+ and Gnao1+/G203R mice in distance traveled (cm) in the open field arena (2-way ANOVA; n.s.). Data are shown as mean ± SEM. Numbers of animals are indicated on bars.
Fig 5.
DigiGait imaging system reveals sex-specific gait abnormalities in Gnao1+/G184S mice and Gnao1+/G203R mice.
(A-D) Female Gnao1+/G184S mice showed significant gait abnormalities, while female Gnao1+/G203R mice remain normal. (A & B) Female Gnao1+/G184S mice showed reduced stride length (2-way ANOVA with Bonferroni multiple comparison post-test) while female Gnao1+/G203R mice were not different from control (2-way ANOVA; n.s.). (C) Female Gnao1+/G184S mice also showed increased paw angle variability (2-way ANOVA, p<0.0001) while female Gnao1+/G203R mice showed normal paw angle variability. (E-H) Male Gnao1+/G203R and Gnao1+/G184S mutant mice showed distinct gait abnormalities. (E & G) Male Gnao1+/G184S mice showed significantly increased paw angle variability (2-way ANOVA p <0.0001 overall with significant Bonferroni multiple comparison tests; **p<0.01 and *p<0.05). There was no effect on stride length. (F & H) In contrast, male Gnao1+/G203R mice showed markedly reduced stride length (2-way ANOVA p<0.0001 with Bonferroni multiple comparison post-test; ***p<0.001, **p<0.01, and *p<0.05) and modestly elevated paw angle variability (overall p<0.05). (I) Gnao1+/G184S mice did not show significant differences in the highest treadmill speed successfully achieved. (J) Both male and female Gnao1+/G203R mice showed reduced capabilities to run on a treadmill at speeds greater than 25 cm/s (Mann-Whitney test; *p<0.05).
Fig 6.
Gnao1+/G203R male mice have an enhanced Pentylenetetrazol (PTZ) kindling response.
(A) Female Gnao1+/G203R mice did not show heightened sensitivity to PTZ injection. (B) Male Gnao1+/G203R mice developed seizures earlier than WT littermates after repeated PTZ injections (Mantel-Cox Test; p<0.05).