Shank3fx is a cre-dependent FLEx switch allele containing an inverted Shank3 PDZ domain (exons 13-16) flanked by inward-facing tandem lox sites (lox2272:loxP). In the absence of Cre recombinase, this functions as a knockout allele and homozygotes exhibit autistic-like characteristics. Subsequent exposure to Cre recombinase that places the Shank3 PDZ domain into the proper orientation for expression results in restored expression of most major SHANK3 isoforms in those cells. These mice allow cre-inducible restoration of SHANK3 expression, and may be useful for studying autism spectrum disorder (ASD), Phelan-McDermid syndrome and schizophrenia.
Guoping Feng, Massachusetts Institute of Technology
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 Shank3fx 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 Shank3fx 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 (Shank3fx/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, Shank3fx/fx mice with widespread expression of tamoxifen-inducible Cre recombinase (Shank3fx/fx:CreER+/-) allow temporal restoration of SHANK3 expression following tamoxifen treatment.
Prior to tamoxifen, they exhibit the same autistic-like phenotype as Shank3fx/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 Shank3fx/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 Shank3fx, 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 Shank3fx 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 Shank3fx 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 Shank3fx allele; resulting in disrupted SHANK3 expression.
The Shank3fx allele was created by Dr. Guoping Feng (Massachusetts Institute of Technology) to have 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. The specific details are below.
The targeting vector was designed with (from 5' to 3') an inward-facing lox2272:loxP site, an inverted Shank3 PDZ domain (exons 13-16), another inward-facing lox2272:loxP site and a frt-flanked neomycin resistance cassette. The targeting vector was electroporated into 129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric males were bred with C57BL/6J females for germline transmission and to establish the colony. Next, the mice were bred with C57BL/6J β-actin Flp mice for germline deletion of the neo cassette. The resulting Shank3fx mice were backcrossed to C57BL/6J inbred mice (Stock No. 000664) for at least two generations. In 2016, Shank3fx males on a mixed C57BL/6J;129S genetic background were sent to The Jackson Laboratory Repository. Coat color was black and agouti. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
|Allele Name||targeted mutation 5.1, Guoping Feng|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Null/Knockout)|
|Gene Symbol and Name||Shank3, SH3 and multiple ankyrin repeat domains 3|
|Strain of Origin||(129X1/SvJ x 129S1/Sv)F1-Kitl+|
|Molecular Note||The targeting vector was designed with (from 5' to 3') an inward-facing lox2272:loxP site, an inverted Shank3 PDZ domain (exons 13-16), another inward-facing lox2272:loxP site and an FRT-flanked neomycin resistance cassette all replacing the corresponding endogenous Shank3 sequences. Flp-mediated recombination removed the FRT-flanked neo cassette.|
Homozygous mice are viable and fertile, with several autistic-like characteristics. When maintaining a live colony, heterozygous mice may be bred together, to wildtype littermates, or to C57BL/6J inbred mice (Stock No. 000664).
When using the Shank3fx (FLexed PDZ domain inverted) mouse strain in a publication, please cite the originating article(s) and include JAX stock #028800 in your Materials and Methods section.
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