SHANK3 is a synaptic scaffolding protein, expressed in the postsynaptic density (PSD) of excitatory synapses. SHANK3 mutations have been identified in cases of intellectual disability such as autism spectrum disorder (ASD), Phelan-McDermid syndrome and schizophrenia.

The Shank3^fx allele has an inverted Shank3 PDZ domain (exons 13-16) that is flanked with the FLEx cassette (inward-facing lox2272:loxP sites), all replacing the corresponding endogenous Shank3 sequences. Prior to Cre recombinase exposure, the Shank3^fx allele functions as a knockout. No protein expression of most major SHANK3 isoforms (including the putative α, β and γ forms) is observed from this allele in striatal postsynaptic density. Homozygous mice (Shank3^fx/fx) recapitulate several autistic-like characteristics, including significant deficits in exploratory behavior, anxiety and motor coordination. The presence of open wound lesions is also suggestive of repetitive grooming behavior. In addition, they show impaired neurotransmission in the dorsal striatum.
When exposure to Cre recombinase places the Shank3 PDZ domain into the proper orientation for expression, the resulting mice have restored expression of most major SHANK3 isoforms (with the exception of the putative SHANK3 γ isoform).

Specifically, Shank3^fx/fx mice with widespread expression of tamoxifen-inducible Cre recombinase (Shank3^fx/fx:CreER^+/-) allow temporal restoration of SHANK3 expression following tamoxifen treatment.
Prior to tamoxifen, they exhibit the same autistic-like phenotype as Shank3^fx/fx mice.
Tamoxifen induction as adult mice (2-4.5 months) rescued the repetitive grooming behavior and social interaction deficit, as well as improved synaptic protein composition, spine density and neural function in the striatum. Adult restoration did not rescue anxiety or motor coordination deficits.

Earlier cre-induced restoration of SHANK3 expression yielded more behavioral improvement than adult treatment. When Shank3^fx/fx mice are bred to have constitutive, widespread cre expression staring at the germ-cell stages (β-actin Cre mice), restoration of SHANK3 rescues the behavioral abnormal behavioral phenotype (normal striatal physiology, exploratory behavior, anxiety and motor coordination).

The two lox variants, lox2272 and loxP, are compatible only with a lox sequence identical to self; they do not recombine with each other. Based on the design of the Shank3^fx, several cre-mediated recombination outcomes are possible based on which lox sites are recombined. The outcomes are described below. In addition, because inward-facing lox sequences result in cre-mediated inversion rather than excision, cre-expressing cells may be able to reversibly switch back-and-forth from the SHANK3-expressing orientations (outcomes i and ii, below) and original knockout orientation as long as Cre recombinase is active in the cell.


i. Inversion of the Shank3^fx allele at the lox2272 sites places the Shank3 PZD domain sequence into the proper orientation for restored SHANK3 expression in the cre-expressing cell type. The Shank3 PZD domain is still flanked by inward-facing lox2272 sites, but the 3' lox2272 site is itself flanked by loxP sites now in the same direction.


ii. Inversion of the Shank3^fx allele at the loxP sites places the Shank3 PZD domain sequence into the proper orientation for restored SHANK3 expression in the cre-expressing cell type. The Shank3 PZD domain is still flanked by inward-facing loxP sites, but the 5' loxP site is itself flanked by lox2272 sites now in the same direction.


iii. Recombination of outcome i at the loxP sites now deletes the 3' lox2272 site, without interruption of SHANK3 expression. This results in permanent SHANK3 expression because the PDZ domain is now flanked with non-compatible lox sites.


iv. Recombination of outcome ii at the lox2272 sites now deletes the 5' loxP site, without interruption of SHANK3 expression. This results in permanent SHANK3 expression because the PDZ domain is now flanked with non-compatible lox sites.


v. Both the inversion of outcome i at the inward-facing lox2272 sites, as well as the inversion of outcome ii at the inward-facing loxP sites reverts to the original Shank3^fx allele; resulting in disrupted SHANK3 expression.