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.
