B6J.Cg-Gad2^tm2(cre)Zjh/MwarJ

Stock number: 028867
Product name: Gad2-IRES-Cre knock-in (C57BL/6J)
Research areas: Neurobiology Research, Research Tools

Description

Gad2-IRES-Cre knock-in mice have Cre recombinase expression directed to GAD2 positive neurons. These mice may be used to generate conditional mutations for studying GAD2 cell populations.
Of note, the same Gad2-IRES-Cre knock-in allele is also available on a C57BL/6N-congenic (Stock No. 019022).

Detailed description

The Gad2-IRES-Cre knock-in allele has an internal ribosome entry site and Cre recombinase in the 3' UTR of the glutamic acid decarboxylase 2 locus (Gad2). As such, the endogenous Gad2 promoter/enhancer elements direct cre expression to GAD2 positive neurons. When Gad2-IRES-Cre mice are bred with mice containing loxP-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in the Gad2-expressing cells (GAD2 positive neurons) in the offspring. Gad2 expression from the Gad2-IRES-Cre allele has not been evaluated. Additional phenotype information described below.

For characterization information of the Gad2-IRES-Cre knock-in allele, see images at the Allen Institute for Brain Science website (Gad2-IRES-Cre images).

If the recombinase activity pattern of this allele is further characterized by the Genetic Resource Science group at The Jackson Laboratory, such findings will be reported on the Mouse Genome Informatics (MGI) Allele Detail entry (Gad2^tm2(cre)Zjh). This same information would also be found searching the MGI Recombinase Activity database.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. The phenotype summarized above is for the parental line: Gad2-IRES-Cre knock-in mice on a mostly C57BL/6;129S4 genetic background (Stock No. 010802). It should be noted that the phenotype of this C57BL/6J-congenic line (Stock No. 028867 Gad2-IRES-Cre knock-in (C57BL/6J)) could vary from that originally described for mice on other genetic backgrounds. We will modify the strain description if necessary as published results become available.

Development

The Gad2-IRES-Cre knock-in allele was designed by Dr. Z. Josh Huang (Cold Spring Harbor Laboratory). A targeting vector was designed to insert an internal ribosome entry site (IRES), a Cre recombinase sequence, a polyA sequence, and an frt-flanked neo cassette into the 3' untranslated region (after the translational termination site) of the glutamic acid decarboxylase 2 locus (Gad2) on chromosome 2. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient tetraploid blastocysts. Chimeric mice were bred with Actin-FLPe mice (on a C57BL/6 congenic background (N10); see Stock No. 005703) to generate the colony and remove the neo selection cassette. These Gad2-IRES-Cre mice were subsequently bred together for many generations (and the FLPe transgene was removed). In 2010, Gad2-IRES-Cre knock-in mice on a mixed genetic background (mostly C57BL/6;129S4 ; with black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. An aliquot of that frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664) and rederive our live colony as Stock No. 010802, which was thereafter maintained by breeding mutant mice together.

In 2015, Dr. Melissa R. Warden (Cornell University) purchased some Stock No. 010802 animals (total backcross generation N1 onto C57BL/6J). They further backcrossed them to C57BL/6J inbred mice for four additional generations. The resulting C57BL/6J-congenic strain, called Gad2-IRES-Cre knock-in (C57BL/6J), were sent to The Jackson Laboratory Repository in 2016 as Stock No. 028867. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664).

In 2017, a 153 SNP analysis (with markers covering all 19 chromosomes and the X chromosome) was performed on the first generation of rederived mice at The Jackson Laboratory Repository (heterozygous for the Gad2-IRES-Cre allele). This showed all markers as C57BL/6J allele-type - the only exceptions being the closest markers flanking the targeted Gad2 locus were segregating with the Gad2-IRES-Cre allele (i.e., heterozygous for C57BL/6J;129 allele-type).

Of note, Dr. Warden's lab 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).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 rederived living colony at The Jackson Laboratory Repository. One of the 43 markers, on Chromosome 2, was segregating.

Allele

Symbol: Gad2^tm2(cre)Zjh
Name: targeted mutation 2, Z Josh Huang
MGI accession ID: MGI:4418713
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Gad2^Cre; Gad2-iCre; Gad2-ires-Cre; GAD65-Cre; GAD65-iCre; GAD65-ires-Cre
Marker symbol: Gad2
Marker name: glutamic acid decarboxylase 2
Marker synonyms: 6330404F12Rik; GAD(65); Gad-2; GAD65
Chromosome: 2
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: When Gad2-IRES-Cre mice are bred with mice containing loxP-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in the Gad2-expressing cells (GAD2 positive neurons) of the offspring.
Molecular note: A targeting vector was designed to insert an internal ribosome entry site (IRES), a Cre recombinase sequence, an SV40 polyA signal, and an frt-flanked neo cassette into the 3' untranslated region (after the translational termination site) of the Gad2 (glutamic acid decarboxylase 2) gene. This construct was electroporated into C57BL6/S129 hybrid embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient tetraploid blastocysts. Chimeric mice were bred with Actin-FLPe mice (on a C57BL/6 congenic background (N10)) to remove the selection cassette. The FLPe transgene was subsequently bred out of the background.