B6N.129P2(Cg)-Igs13^tm1Dolm Igs14^tm1Dolm/J

Stock number: 025330
Product name: 16p11.2^flx
Strain synonyms: 16p11^flx
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools, Sensorineural Research

Description

A copy number variation on human chromosome 16p11.2 is among the most common genetic variations found in autism spectrum disorders. 16p11^flx mice are a Cre or FLP recombinase-inducible mouse model of 16p11.2 deletion that has several loxP and frt sites flanking the corresponding 440 kbp region on mouse chromosome 7F3. These mice may be useful in studying basal ganglia circuitry and the pathophysiology of autism.

Detailed description

A copy number variation (CNV) on human chromosome 16p11.2 is among the most common genetic variations found in autism spectrum disorders (ASD). Patients with this deletion display motor deficits, speech/language delay, and cognitive impairments, accompanied by ASD, attention deficit hyperactivity disorder (ADHD), seizures, and hearing disorders. Conversely, a duplication of 16p11.2 is associated with schizophrenia. The chromosome 16p11.2 CNV encompasses 26 genes that are highly conserved on mouse chromosome 7F3.

16p11^flx is a Cre or FLP recombinase-inducible mouse model of 16p11.2 deletion that has several loxP and frt sites flanking the 440 kbp region on mouse chromosome 7F3. Homozygous mice (16p11^flx) are viable and fertile with no reported gross physical or behavioral abnormalities.

Following Cre or FLP-mediated deletion of the entire region, a membrane-targeted fluorescent reporter gene (mCherry) is expressed. The donating investigator reports that for imaging large-scale anatomical structures, such as white matter tracts, mCherry fluorescence is very strong. For identification of single neuronal cell-bodies, however, immunohistochemical fluorescence might improve the outcome.

Mice with deletion of the genomic segment (16p11^-) are available at The Jackson Laboratory as Stock No. 025100.

The 16p11^flx model was created using two independent targeting events. Although the loxP/frt region near Coro1a is in relatively close proximity (440 kbp) to the loxP/frt region near Spn, the two loci have a small chance of segregating independently of one another. The donating investigator reports they never observed any crossing over between the two loci and they always segregated together.

Additionally, breeding heterozygous mice together at The Jackson Laboratory results in mice that are black, light chinchilla or albino. This suggests the 16p11^flx mice we received retained mESC-derived allele(s) at the tyrosinase locus (e.g., Tyr^c and/or Tyr^c-ch) that, because it is only ~40 kbp proximal on chromosome 7, appears to be segregating along with the 16p11^flx region. Depending upon future segregation patterns, available mice may be black, light chinchilla or albino.

Development

16p11^flx has several loxP and frt sites flanking the 440 kbp region on mouse chromosome 7F3; a region highly conserved with the human chromosome 16p11.2 region whose copy number variations are associated with autism spectrum disorders and schizophrenia.

Drs. Ricardo E. Dolmetsch and Thomas Portmann (while at Stanford University) created the two targeting vectors used to generate these 16p11^flx mice. The first vector was designed to insert a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), loxP-frt-neomycin resistance cassette-loxP-frt-loxP sequence and polyA signal at the centromeric end of the coronin actin binding protein 1A locus (Coro1a). The insertion site was 1165 bp centromeric (transcriptionally downstream) of the last Coro1a exon to avoid disrupting endogenous expression prior to Cre recombination. The second targeting vector was designed to insert a translational stop codon (for all frames), loxP-frt-puromycin resistance cassette-loxP-frt sequence, rabbit b-globin intron, coding sequence mCherry fluorescent protein (with palmitoylation signal for membrane targeting) and polyA signal at the telomeric end of the sialophorin locus (Spn). The insertion site was 3336 bp telomeric (transcriptionally upstream) of the Spn gene to avoid disrupting endogenous expression prior to Cre recombination. Sequential targeting into 129 mouse embryonic stem cells (mESC [see note below]) resulted in mESC clones with cis arrangement (targeting into the same chromosome 7 homolog) or trans arrangement (each vector targeted into different chromosome 7 homologs). The modified mESCs were injected into blastocysts, implanted into pseudopregnant female mice, and chimeric males were subsequently bred to C57BL/6NCrl females and/or HPRT-Cre females (Hprt^tm1(cre)Mnn on a C57BL/6;CD1 mixed genetic background; see Stock No. 004302). Germline transmission was successful only for the cis arrangement, and animals with no recombination resulted in heterozygous floxed mice (16p11^flx/+). The 16p11^flx colony was bred to C57BL/6NCrl for at least five generations prior to sending black males to The Jackson Laboratory in 2014. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (from Stock No. 005304).

Additionally, breeding heterozygous mice together at The Jackson Laboratory results in mice that are black, light chinchilla or albino. While the Portmann et al. 2014 Cell Rep 7(4):1077-92 publication describes the mESC used were 129/OLA, the donating laboratory indicates it may have been a different 129 origin. Taken together, these data suggest the 16p11^flx mice we received retained mESC-derived allele(s) at the tyrosinase locus (e.g., Tyr^c and/or Tyr^c-ch) that, because it is only ~40 kbp proximal on chromosome 7, appears to be segregating along with the 16p11^flx region. Depending upon future segregation patterns, available mice may be black, light chinchilla or albino.

In 2015, a 32 SNP panel analysis (27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains), was performed on the first generation rederived live colony at The Jackson Laboratory Repository. This revealed one animal with 2 of 27 markers, one each on chromosomes 2 and 12, that were not fixed for C57BL/6 allele-type (e.g., still segregating for 129 allele-type markers). All 5 markers that determine C57BL/6 substrains were found to be C57BL/6N allele-type.

Allele

Symbol: Igs14^tm1Dolm
Name: targeted mutation 1, Ricardo Dolmetsch
MGI accession ID: MGI:5569503
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; No functional change
Marker symbol: Igs14
Marker name: intergenic site 14
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: Following Cre or FLP-mediated deletion of the 440 kbp region on mouse chromosome 7F3region, a membrane-targeted fluorescent reporter gene (mCherry) is expressed in Cre or FLP expressing tissues.
Molecular note: The targeting vector is designed to insert a translational stop codon (for all frames), loxP-frt-puromycin resistance cassette-loxP-frt sequence, rabbit b-globin intron, coding sequence mCherry fluorescent protein (with palmitoylation signal for membrane targeting) and polyA signal at the telomeric end of the sialophorin locus (Spn) gene. The insertion site is 3336 bp telomeric (transcriptionally upstream).

Allele

Symbol: Igs13^tm1Dolm
Name: targeted mutation 1, Ricardo Dolmetsch
MGI accession ID: MGI:5569502
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Igs13
Marker name: intergenic site 13
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Site of expression: Following Cre or FLP-mediated deletion of the 440 kbp region on mouse chromosome 7F3region, a membrane-targeted fluorescent reporter gene (mCherry) is expressed in Cre or FLP expressing tissues.
Molecular note: The targeting vector is designed to insert a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), loxP-frt-neomycin resistance cassette-loxP-frt-loxP sequence and polyA signal at the centromeric end of the coronin actin binding protein 1A locus (Coro1a) gene. The insertion site is 1165 bp centromeric (transcriptionally downstream) of the last exon.