B6.Cg-Cacna1c^tm2Itl/J

Stock number: 019547
Product name: TS2-neo
Strain synonyms: G406R
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research

Description

The TS2-neo mutation has the G406R mutation associated with severe Timothy Syndrome (TS2) and an inverted neomycin resistance cassette, all inserted at the end of exon 8 of the CaV1.2 L-type calcium channel locus (Cacna1c). Heterozygous TS2-neo mice exhibit several characteristics of autism spectrum disorder, including repetitive/perseverative stereotypical behavior, decreased social interaction, decreased communication/vocalization, enhanced fear memory and self-injurious scratching. These mice are useful in studying the autism spectrum disorder (ASD) characteristics of Timothy Syndrome.

Detailed description

The TS2-neo allele has a G406R mutation associated with severe Timothy Syndrome (TS2) and an inverted neomycin resistance cassette, all inserted at the end of exon 8 of the CaV1.2 L-type calcium channel locus (Cacna1c). In similar G406R mutant mice without a neo cassette, expression of the G406R mutation-bearing channel causes death in both heterozygotes and homozygotes. Mice homozygous for the TS2-neo allele are also lethal. However, a single copy of the TS2-neo allele results in 75% lower G406R mutation-bearing channel expression in brain and no expression of the G406R mutation-bearing channel in heart (suggesting transcriptional interference from the neomycin cassette). The attenuated expression diminishes the deleterious effects of mutant L-type channel overactivity and allows heterozygous TS2-neo animals to be viable and fertile. Heterozygous TS2-neo mice exhibit distinct traits strikingly reminiscent of the entire core triad of autism spectrum disorder (ASD): repetitive/perseverative behavior, impaired social behavior, and impaired communication. TS2-neo mice also display enhanced fear memory (reminiscent of persistent memories or abnormal fear associated with ASD), as well as self-injurious scratching.

Development

The TS2-neo mutation was designed by Dr. Randall L. Rasmusson (The State University of New York, University at Buffalo). First, a G->A base pair substitution was introduced (via site-directed mutagenesis) at the end of exon 8 of the CaV1.2 L-type calcium channel locus (Cacna1c) on chromosome 6. This mutation at the intracellular part of the S6 transmembrane segment of domain 1 results in a de novo gain of function missense substitution of glycine to arginine at codon 406 (G406R); a mutation associated with severe Timothy Syndrome (TS2). The targeting vector was designed to introduce this G406R mutation at the end of exon 8, as well as an inverted neo cassette (frt::loxP::inverted neomycin resistance sequence::frt::loxP) 301 bp downstream of the G->A point mutation, thus causing the introduction of a stop codon in exon 8A. The targeting vector was electroporated into (C57BL/6NTac x 129S6/SvEvTac)F1-derived iTL BA1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred with unknown animals to establish the mutant colony. TS2-neo mice were then backcrossed with C57BL/6J for at least nine generations prior to sending to The Jackson Laboratory Repository in 2013. Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). Heterozygous TS2-neo mice were then bred to wildtype mice from the colony or with C57BL/6J inbred mice to maintain the colony. The donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Cacna1c^tm2Itl
Name: targeted mutation 2, inGenious Targeting Laboratory
MGI accession ID: MGI:5296904
Generation method: Targeted
Attributes: Null/Knockout; Humanized sequence
Marker symbol: Cacna1c
Marker name: calcium channel, voltage-dependent, L type, alpha 1C subunit
Chromosome: 6
Strain of origin: (C57BL/6NTac x 129S6/SvEvTac)F1
Molecular note: A G to A transition at the end of exon 8 results in a G406R mutation which appears to be a gain of function mutation that is embryonic lethal in both hetero- and homozygotes. The insertion of an FRT:loxP flanked-inverted Neo cassette 301 bp downstream of the G->A point mutation introduces a stop codon in alternative exon 8A (which is mutually exclusive with exon 8) and ameliorates the lethality of the point mutation in heterozygotes.