B6;129-Esyt1^tm1Sud Esyt2^tm1Sud/J

Stock number: 027583
Product name: 12EN ESYT1/ESYT2 conditional KO
Research areas: Cell Biology Research, Research Tools

Description

Esyt1/Esyt2 conditional mice possess recombination cassettes that allow for the development of different conditional, KO, and KI lines. These mice are useful when studying Ca²⁺ signaling at endoplasmic reticulum-plasma membrane junctions.

Detailed description

Esyt1/Esyt2 conditional mice possess recombination cassettes that allow for the development of different conditional and KO lines. Esyt1 and Esyt2 encode membrane proteins that are involved with the formation of junctions between endoplasmic reticulum (ER) and plasma membrane (PM), as well as the Ca²⁺ signaling and lipid trafficking at ER-PM junctions. Each allele contains a mutation in exon 10 that changes four aspartate residues into alanine residues (D4A). The aspartate residues are required for calcium binding of the ESYT1 and ESYT2 proteins, therefore when mutated into alanines the proteins should not be able to bind calcium. Mice that are homozygous for both alleles are viable and fertile.

When bred to mice expressing FLP Recombinase, recombination of frt sites should result in a strain with loxP sites flanking exon 10 of the Esyt1 and Esyt2 genes. Subsequent breeding to a strain expressing Cre Recombinase can be expected to result in offspring with the floxed exons deleted, creating a double KO strain.

When bred to a mouse expressing FLP Recombinase, recombination of F3 sites is designed to remove the endogenous exons 10 of both Esyt1 and Esyt2 genes, and only express the mutated D4A version of each exon.

Development

A targeting vector was designed to insert an F3 site followed by an attB site and a loxP site upstream of exon 10 of the extended synaptotagmin-like protein 1 (Esyt1) gene. Inserted downstream of exon 10, from 5’ to 3’, are a second loxP site, an attP site, a frt site, a neomycin resistance cassette, a second F3 site, and a mutated exon 10 with four aspartate residues mutated into alanine residues (D4A) followed by a second frt site. The construct was electroporated into C57BL/6-derived B6-3 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred to C57BL/6 mice.

Another targeting vector was designed to insert an F3 site followed by a loxP site upstream of exon 10 of the extended synaptotagmin-like protein 2 (Esyt2) gene. Inserted downstream of exon 10, from 5’ to 3’, are a second loxP site, a frt site, a neomycin resistance cassette, a second F3 site, a loxP2272 site, and a mutated exon 10 with four aspartate residues mutated into alanine residues (D4A) followed by a second loxP2272 site and a second frt site. The construct was electroporated into C57BL/6-derived B6-3 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred to C57BL/6 mice.

Mice from each line were bred together to generate these double conditional mice. Mice were maintained on a mixed B6;129sv background. Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Esyt2^tm1Sud
Name: targeted mutation 1, Thomas C Sudhof
MGI accession ID: MGI:5636380
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Esyt2
Marker name: extended synaptotagmin-like protein 2
Chromosome: 12
Strain of origin: C57BL/6NTac
Molecular note: A targeting vector is designed to insert an F3 site followed by a loxP site upstream of exon 10. Inserted downstream of exon 10, from 5' to 3' , are a second loxP site, a FRT site, a neomycin resistance cassette, a second F3 site, a loxP2272 site, and a mutated exon 10 with four aspartate residues mutated into alanine residues (D4A) followed by a second loxP2272 site and a second FRT site.

Allele

Symbol: Esyt1^tm1Sud
Name: targeted mutation 1, Thomas C Sudhof
MGI accession ID: MGI:5636379
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Esyt1
Marker name: extended synaptotagmin-like protein 1
Chromosome: 10
Strain of origin: C57BL/6NTac
Molecular note: A targeting vector was designed to insert an F3 site followed by an attB site and a loxP site upstream of exon 10. Inserted downstream of exon 10, from 5'to 3' are a second loxP site, an attP site, a FRT site, a neomycin resistance cassette, a second F3 site, and a mutated exon 10 with four aspartate residues mutated into alanine residues (D4A) followed by a second FRT site.