STOCK Shank3^tm3.1Gfng/J

Stock number: 028778
Product name: InsG3680 Shank3 (Shank3*G3680 knock-in)
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

The InsG3680 Shank3 knock-in allele (Shank3*InsG3680) harbors the autism spectrum disorder (ASD)-associated InsG3680 mutation; a single guanine nucleotide insertion at Shank3 cDNA position 3680 that creates a frameshift and downstream stop codon. Homozygous mice lack expression of major SHANK3 protein isoforms; resulting in synaptic and behavioral characteristics similar to human ASD, in both juvenile and adult.

Detailed description

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 InsG3680 Shank3 knock-in allele harbors the ASD-associated InsG3680 mutation; a single guanine nucleotide insertion at Shank3 cDNA position 3680 that creates a frameshift and downstream stop codon. While the predicted size of the InsG3680 truncated protein is 135 kDa, the InsG3680 mutation results in loss of major SHANK3 protein isoforms.

InsG3680 Shank3 mice on a mixed C57BL/6J;129S genetic background were characterized in the original publication (Zhou et al. 2016 Neuron 89:147); and this is described below.

The donating investigator reports that mice homozygous for the InsG3680 Shank3 knock-in allele (InsG3680^+/+) are viable and fertile, but are inefficient breeders. Homozygotes exhibit synaptic and behavioral phenotypes similar to human ASD. Specifically, InsG3680^+/+ manifest striatal synaptic transmission defects before weaning age and impaired juvenile social interaction; coinciding with the early onset (before three years of age) of symptoms observed in human ASD. Adult InsG3680^+/+ display increased anxiety-like behavior, reduced social interaction and grooming-induced self-injurious lesions. Additionally, homozygous cortex shows SHANK1/2 upregulation compared to wildtype cortex. Heterozygous mice (InsG3680^+/-) have ~50% of the wildtype levels of full-length SHANK3 protein, and exhibit a less severe phenotype.

Development

The InsG3680 Shank3 knock-in allele (Shank3*InsG3680) was designed by Dr. Guoping Feng (Massachusetts Institute of Technology) to alter exon 21 of the SH3/ankyrin domain gene 3 (Shank3) on chromosome 15 by inserting a single guanine nucleotide at cDNA position 3680; causing a frameshift and downstream stop codon. This InsG3680 mutation was first identified in two brothers diagnosed with autism spectrum disorder (ASD) accompanied by severe mental retardation. A loxP-flanked PGK-neo-pA cassette was also inserted downstream of the mutant stop codon (but was later removed as described below). The targeting vector was co-electroporated with a pair of TALE nuclease plasmids (designed to facilitate recombination) into 129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred with C57BL/6J for germline transmission and to establish the colony. The PGK-neo-pA cassette was deleted by breeding to C57BL/6 mice with germline Cre recombinase expression. The resulting InsG3680 Shank3 mice were backcrossed to C57BL/6J inbred mice (Stock No. 000664) for at least five or six generations (during which the Cre recombinase allele was removed). Next, they were bred to 129S1/SvImJ inbred mice (Stock No. 002448) for at least one generation. InsG3680 Shank3 mice on this mixed C57BL/6J;129S genetic background were characterized in the original publication (Zhou et al. 2016 Neuron 89:147), and males were sent to The Jackson Laboratory Repository in 2016. 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 B6129SF1 oocytes (Stock No. 101043).

Allele

Symbol: Shank3^tm3.1Gfng
Name: targeted mutation 3.1, Guoping Feng
MGI accession ID: MGI:5775620
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Shank3
Marker name: SH3 and multiple ankyrin repeat domains 3
Chromosome: 15
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: The allele was designed to alter exon 21 by inserting a single guanine nucleotide at cDNA position 3680; causing a frameshift and downstream stop codon. A loxP-flanked PGK-neo-pA cassette was also inserted downstream of the mutant stop codon. Cre-mediated recombination removed the floxed neo cassette. The mutation was first identified in two brothers diagnosed with autism spectrum disorder (ASD) accompanied by severe mental retardation. Antibodies to both the N and C terminus do not detect protein in striatal tissue.