B6;129S2-Syngap1^tm2Geno/RumbJ

Stock number: 029304
Product name: SYNGAP1^lx-st (Cre-conditional rescue)
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

The SYNGAP1^lx-st conditional rescue knock-in allele has a loxP-flanked neo-STOP cassette upstream of exon 6 of the synaptic RasGAP (SynGAP) gene; resulting in disrupted expression of full-length SynGAP and expression of a truncated/inactive SynGAP protein. These mice allow Cre recombinase-inducible restoration of full-length SynGAP expression. SYNGAP1^lx-st mice may be useful Cre-lox studies of synapse development (specifically in dendritic spine), cognitive and behavioral maturation, intellectual disability and autism spectrum disorder.

Detailed description

Mutations that cause intellectual disability (ID) and autism spectrum disorder (ASD) are commonly found in genes that encode for synaptic proteins. Syngap1 encodes a synaptic RasGAP (SynGAP) that is largely localized to dendritic spines in neocortical pyramidal neurons, where it suppresses signaling pathways linked to NMDA receptor (NMDAR)-mediated synaptic plasticity and AMPA receptor (AMPAR) membrane insertion. Syngap1 has alternative transcriptional start sites and several alternatively spliced C-terminal exons that result in many possible SynGAP isoforms. Human SYNGAP1 haploinsufficiency results from a truncation of the full-length protein. Exon 7 contains the first common methionine present in the shortest splice variant.

The SYNGAP1^lx-st allele has a loxP-flanked neo-STOP cassette upstream of exon 6 of the Syngap1 gene. Following cre-mediated recombination that removes the floxed region, SynGAP expression and function are fully recovered.

Prior to Cre recombinase exposure, mice heterozygous for the SYNGAP1^lx-st allele express a SynGAP protein truncated after exon 5 (known to be an inactivate SynGAP protein), as well as diminished levels of functional SynGAP (from the wildtype allele). Therefore, the phenotype of heterozygotes (SYNGAP1^lx-st/+) is the same as mice heterozygous for the global knockout allele, and both are a model of human SYNGAP1 haploinsufficiency - exhibiting normal synaptic transmission, modest defects in synaptic plasticity, enhanced synaptic function (accelerated rate of glutamatergic synapse maturation) during early neural development and profound cognitive and behavioral abnormalities. Specifically, heterozygous SynGAP-deficiency significantly disrupts excitatory/inhibitory (E/I) balance in the neural networks that support cognition and behavior. Abnormal behavioral phenotypes can be observing as early as 21 days of age with ~100% penetrance (although physiological abnormalities can be measured even earlier through other methods such as electrophysiological recordings). Furthermore, homozygotes (SYNGAP1^lx-st/lx-st are phenotypically the same as global knockout homozygotes - exhibiting complete postnatal lethality between 2-5 days of age.

When SYNGAP1^lx-st are bred to mice that express Cre recombinase, the resulting offspring may be useful in generating tissue-specific restoration of full-length SynGAP expression. For example, when SYNGAP1^lx-st/+ mice are bred hemizygous for the CAGGCre-ER^TM transgene (Stock No. 004682), the resulting animals allow widespread, tamoxifen-inducible SynGAP restoration. In those mice, complete restoration of SynGAP protein expression in adults had no detectable benefit on behavior or cognition - demonstrating that SYNGAP1 haploinsufficiency is a disorder of brain development.

Development

The SYNGAP1 conditional rescue mice (SYNGAP1^lx-st) was constructed by genOway S.A. (France) as a pay-for-service on behalf of Dr. Gavin Rumbaugh (The Scripps Research Institute). The targeting vector was designed to insert a loxP-flanked STOP cassette (neomycin resistance gene and a series of stop codons) immediately downstream of exon 5 of the synaptic Ras GTPase activating protein 1 homolog (rat) locus (Syngap1) on chromosome 17.

The construct was electroporated into 129S2/SvPas-derived embryonic stem (ES) cells. Correctly targeted ES cells were identified and injected into recipient blastocysts. Chimeric males were bred to C57BL/6J for germline transmission.

The SYNGAP1^lx-st colony was maintained on this mixed genetic background for several generations prior to sending males (with black or agouti coat color) to The Jackson Laboratory Repository in 2012.

Upon arrival, males were used to cryopreserve sperm. To establish a living mouse colony, an aliquot of the frozen sperm may be used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Syngap1^tm2Geno
Name: targeted mutation 2, Genoway
MGI accession ID: MGI:5469866
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Syngap1
Marker name: synaptic Ras GTPase activating protein 1 homolog (rat)
Chromosome: 17
Strain of origin: 129S2/SvPas
Expressed genes: Syngap1,synaptic Ras GTPase activating protein 1 homolog (rat),mouse, laboratory
Molecular note: A floxed neo cassette was inserted into intron 5. Western blot analysis confirmed reduced protein expression in heterozygous samples. Cre-mediated recombination is required to remove the neo cassette and restore protein expression.