Apparent Genetic Rescue of Adult Shank3 Exon 21 Insertion Mutation Mice Tempered by Appropriate Control Experiments

Tamoxifen treatment strategy. Experimental mice of all six genotypes (Shank3^+/+Cre^Tam−, Shank3^+/GCre^Tam−, Shank3^G/GCre^Tam−, Shank3^+/+Cre^Tam+, Shank3^+/GCre^Tam+, and Shank3^G/GCre^Tam+) were fed vehicle diet until eight weeks of age. At eight weeks, mice were separated into two treatment groups, one receiving vehicle diet for six weeks and another receiving tamoxifen diet for six weeks. Each treatment group consisted of mice from all six genotypes. After the six-week treatment, all mice were fed vehicle diet for at least a two-week wash-out period before testing. During and after testing, all mice were fed vehicle diet.

Tamoxifen-inducible Shank3 genetic reversal strategy. The Shank3^G mutation was introduced into the WT Shank3 allele (A) by insertion of a neo-STOP cassette before WT exon 21 (B). The neo-STOP cassette was flanked by loxP sites, so that Cre-recombinase activity in the nucleus could excise the mutated Shank3^G exon 21 (C), resulting in restoration of the WT Shank3^+/+ gene (D) and SHANK3 expression, thereby effectively reversing the Shank3^G mutation.

Optimization of tamoxifen treatment protocol in adult Shank3^GCre^Tam+ mice. A, Quantification of Western blotting showing minimal rescue of WT SHANK3 protein expression following treatment with 4-hydroxytamoxifen (66.67 mg/kg) given once per day subcutaneously for 15 d (n = 7 for all treatment groups). B, Representative Western blotting (top) and quantification of whole-brain lysates (bottom) showing degrees of rescue of SHANK3 protein expression after varying duration of tamoxifen diet (n = 7 for all treatment groups). C, SHANK3 protein levels from whole-brain lysates in adult Shank3^GCre^Tam− mice treated for six weeks with vehicle or tamoxifen diet (vehicle diet: Shank3^+/+ n = 9, Shank3^+/G n = 7, Shank3^G/G n = 4; tamoxifen diet: Shank3^+/+ n = 12, Shank3^+/G n = 9, Shank3^G/G n = 9). D, SHANK3 protein levels from whole-brain lysates in adult Shank3^GCre^Tam+ mice treated for six weeks with vehicle diet or tamoxifen diet [vehicle diet: Shank3^+/+ n = 26, Shank3^+/G n = 21, Shank3^G/G n = 15; tamoxifen diet: Shank3^+/+ n = 23, Shank3^+/G n = 15, Shank3^G/G n = 18; data are normalized to the β-actin control and then to the average of WT levels with C-terminal SHANK3 antibody (JH3025)]. E, Example Southern blotting of brain tissue in Cre^Tam+, WT and Shank3^G/G homozygous mutant mice treated with vehicle or tamoxifen (TAM; Rev = band expected following cre-mediated recombination of floxed mutant; Mut = band expected for floxed mutant before cre recombination; WT = band expected in WT mice without mutant allele); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent S.E.M. in this and all subsequent figures.

Vehicle-treated Shank3^G/GCre^Tam− mice exhibit clear behavioral and physiologic phenotypes. Shank3^G/GCre^Tam− mice exhibit a novelty avoidance phenotype by building smaller nests in both nest width (A) and height (B) over a 90-min period, burying fewer marbles over a 30-min period (C), and exhibiting hypoactivity within the first 5 min of the open field test (D) compared to Shank3^+/+Cre^Tam− controls. E, Rotarod testing demonstrates that motor learning and coordination are decreased in Shank3^G/GCre^Tam− mice compared to controls. Shank3^+/+ n = 27, Shank3^+/G n = 18, Shank3^G/G n = 19. F, Synaptic transmission in the hippocampus is impaired in Shank3^G/GCre^Tam− mice with decreased fEPSP in response to stimulus intensity compared to Shank3^+/+Cre^Tam− mice. Inset, Average of five consecutive raw traces at stimulus intensities 0–350 μA in 50-μA steps from Shank3^+/+ (top) and Shank3^G/G (bottom) mice; scale bar = 0.5 mV, 5 ms. Shank3^+/+ n = 10 slices from five mice, Shank3^G/G n = 9 slices from three mice; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Behavioral and synaptic phenotypes in Shank3^G/GCre^Tam− mice are not rescued by treatment with six weeks of tamoxifen diet. Nesting behavior is not rescued by tamoxifen treatment in Shank3^G/GCre^Tam− mice with regard to nest width (A), although there is no main effect of genotype with regard to nest height (B). Marble burying (C) remains impaired in tamoxifen-treated Shank3^G/GCre^Tam− mice with decreased number of marbles buried compared to controls. Locomotor activity also remains decreased in tamoxifen-treated Shank3^G/GCre^Tam− mice at the start of the open field test (D). Latency to fall off the rotarod (E) remains decreased in tamoxifen-treated Shank3^G/GCre^Tam− mice compared to controls. Shank3^+/+ n = 25, Shank3^+/G n = 19, Shank3^G/G n = 16. F, Tamoxifen treatment does not rescue synaptic transmission in Shank3^G/GCre^Tam− mice. Inset, Average of five consecutive traces at each stimulus intensity 0–350 μA in 50-μA steps from Shank3^+/+ (top) and Shank3^G/G (bottom) mice treated with six weeks of tamoxifen diet; scale bar = 0.25 mV, 5 ms. Shank3^+/+ n = 22 slices from eight mice, Shank3^G/G n = 20 slices from six mice; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Incomplete genetic rescue of behavioral and physiologic phenotypes in tamoxifen-treated Shank3^G/GCre^Tam+ mice. There is no main effect of genotype on nest width (A) or height (B) in tamoxifen-treated Shank3^G/GCre^Tam+ mice, suggesting successful rescue of the Shank3^G/G nest-building phenotype. C, The Shank3^G/G marble-burying phenotype is not rescued in tamoxifen-treated Shank3^G/GCre^Tam+ mice, nor is the initial hypoactivity observed in the open field test (D). Tamoxifen treatment of Shank3^G/GCre^Tam+ mice does successfully eliminate the main effects of genotype in the time to fall from the rotarod. Shank3^+/+ n = 23, Shank3^+/G n = 15, Shank3^G/G n = 18 (E). F, There is no main effect of genotype on fEPSP slope over a range of stimulus intensities in tamoxifen-treated Shank3^+/+Cre^Tam+ and Shank3^G/GCre^Tam+ mice. Inset, Average of five consecutive traces at each stimulus intensity 0–350 μA in 50-μA steps from Shank3^+/+ (top) and Shank3^G/G (bottom) mice treated with six weeks of tamoxifen diet; scale bar = 0.25 mV, 5 ms. Shank3^+/+ n = 16 slices from eight mice, Shank3^G/G n = 12 slices from six mice; *p < 0.05, ***p < 0.0001, ****p < 0.00001.

Unexpected, partial genetic “rescue” of behavioral and synaptic phenotypes in vehicle-treated Shank3^G/GCre^Tam+ mice. The decreases in nest width (A) and height (B) in Shank3^G/G mice are rescued in vehicle-treated Shank3^G/GCre^Tam+ mice. The Shank3^G/G marble-burying phenotype (C) and initial locomotor hypoactivity in the open field test (D) are not rescued in vehicle-treated Shank3^G/GCre^Tam+ mice. E, There is no main effect of genotype in time to fall from the rotarod in vehicle-treated Shank3^GCre^Tam+ mice. Shank3^+/+ n = 26, Shank3^+/G n = 21, Shank3^G/G n = 15. F, There is no main effect of genotype on fEPSP slope in response to a range of stimulus intensities. Inset, Average of five consecutive traces at each stimulus intensity 0–350 μA in 50-μA steps from Shank3^+/+ (top) and Shank3^G/G (bottom) mice treated with vehicle diet; scale bar = 0.25 mV, 5 ms. Shank3^+/+ n = 16 slices from six mice, Shank3^G/G n = 16 slices from five mice; *p < 0.05, ***p < 0.0001, ****p < 0.00001.