B6.129(Cg)-Shank3^em1Yhj/J

Stock number: 039525
Product name: Shank3e4-22 flox
Research areas: Neurobiology Research

Description

This Shank3e4-22 flox conditional knock-out strain has two loxP sequences flanking the protein-coding exons (exons 4 through 22). The first loxP is in between exon 3 and 4 and the second is inserted after the terminal cording codon exon 22. Homozygous mice are normal before Cre recombination to induce exons4-22 deletion. These mice may be useful for studying behavioral abnormalities associated with Autism Spectrum Disorder (ASD) and Phelan-McDermid syndrome.

Detailed description

The targeted Shank3 gene encodes a synaptic scaffolding protein, expressed in the postsynaptic density of excitatory synapses. Mutations in this gene have been associated with autism spectrum disorders and schizophrenia. These Shank3e4-22 flox mice have two loxP sites inserted into upstream of exon 4 and after the final coding exon 22. When crossed to cre-expressing mice, a full KO exons 4-22 deletion can result. Homozygotes are viable and fertile. These mice may be useful for studying behavioral abnormalities associated with Autism Spectrum Disorder (ASD) and Phelan-McDermid syndrome.

Of note, the donating investigator has donated Shank3Δe4-22 full knock-out mice (Stock No. 039524) and Shank3^e4-9 knock-in/knock-out mice (Stock No. 017442).

Development

The Shank3e4-22 flox allele was generated by a two-step targeting strategy using the Cre-loxP system, using a targeting vector containing an FRT site flanked Neo selection cassette and an FRT site flanked Puro selection cassette. The first (5') construct inserted a loxP site upstream of exon 4 and a FRT-Neo-FRT-loxP cassette upstream of exon 10 of the targeted gene. The second (3') construct inserted a FRT-Puro-FRT-loxP cassette ~5 kb downstream of exon 22 of the targeted gene. These constructs were sequentially electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were tested for germline transmission and crossed to a C57BL/6 mouse line to generate the Shank3^tm2.1Yhj allele having 3 loxP sites. Then using CRISPR/Cas9 methodologies, a sgRNA was designed to target the intron 9 loxP site. After pronuclear injection of the Cas9 and sgRNA vector, PCR and genomic sequencing were done on the founder mouse to confirm a 109 bp deletion (including the intron 9 loxP) and the intactness of the loxP site upstream of exon 4 and loxP site downstream of exon 22. The founder was then bred with a C57BL/6 mouse to generate heterozygous offspring and further backcrossed to C57BL/6 for more than 5 generations. The heterozygotes breeding was used to maintain the line and generate the homozygous mice. After another ~10 generations of breeding, homozygous breeding was used to maintain the colony in the donating laboratory. The 129 inbred strain Disc1^del allele variant was segregated away from the Shank3e4-22 flox allele during backcrossing. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Shank3^em1Yhj
Name: endonuclease-mediated mutation 1, Yong-hui Jiang
MGI accession ID: MGI:7738562
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Shank3
Marker name: SH3 and multiple ankyrin repeat domains 3
Marker synonyms: DEL22q13.3; PROSAP2; PSAP2; SCZD15; SPANK-2
Chromosome: 15
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: A loxP site upstream of exon 4 and a second loxP site upstream of exon 10 of the Shank3 gene flanking exons 4-9 and the third loxP site 5-kb downstream of exon 22 generated in Shank3^tm2.1Yhj was targetted by CRISPR/cas9 methodologies to remove the second loxP site. Only two loxP sites flanking exons 4-22 of the Shank3 gene remain, one loxp between exon 3 and exon 4, and another loxp after exon 22.