B6.Cg-Gad2^tm1Rpa Gad1^tm1Rpa Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/RpaJ

Stock number: 031800
Product name: Gad1^lox::Gad2^lox::Rosa26^tdTomato
Research areas: Neurobiology Research, Research Tools

Description

This triple mutation strain incorporates floxed alleles of both the mouse Gad1 and Gad2 genes in addition to a conditional floxed-Stop Gt(ROSA)26Sor-tdTomato reporter. Cre-mediated excision of the floxed regions in Gad1 and Gad2 creates knock-out alleles of the two genes, and simultaneously activates cre-specific tdTomato reporter expression. Gad1 (GAD67) and Gad2 (GAD65) are essential for GABA biosynthesis.

Detailed description

Gad1 (glutamate decarboxylase 1; also called GAD67) and Gad2 (glutamate decarboxylase 2; also called GAD65) encode enzymes essential for GABA biosynthesis.

This triple mutation strain incorporates floxed alleles of both the mouse Gad1 and Gad2 genes in addition to a conditional floxed-Stop Gt(ROSA)26Sor-tdTomato reporter. Cre-mediated excision of the floxed regions in Gad1 and Gad2 creates knock-out alleles of the two genes, simultaneously activating cre-specific tdTomato reporter expression.

Functionality of this strain was demonstrated by intercrossing these mice with animals expressing aminoglycoside (NB124)-inducible Agrp^ns-Cre (agouti related neuropeptide, nonsense suppressor cre). Co-localization of tdTomato marker and AGRP peptide was observed in the ARC region of the hypothalamus 4 days after injection of NB124 into the third ventricle of the brain. GABA protein expression was abolished (as determined by immunostaining) and RNA was almost completely depleted. GABA synthesis was effectively disrupted in AGRP-expressing neurons.

Development

To create the Gad1^lox mutation: a targeting vector was used to introduce a loxP site in intron 1 and a loxP-FRT-SV-neomycin-FRT cassette to intron 2 of the mouse Gad1 gene via homologous recombination in (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. The FRT-flanked neomycin cassette was excised from resultant mice via crosses with Flp recombinase-expressing mice, leaving exon 2 (the first coding exon) flanked by loxP sites.

To create the Gad2^loxP allele: exon 1 of the mouse Gad2 gene was flanked by loxP sites and an FRT-flanked SV-neomycin cassette was placed in intron 1 via homologous recombination in (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. The SV-neomycin cassette was retained in this mutation.

Mice carrying the above Gad1^lox and Gad2^lox mutations were intercrossed with floxed-Stop Gt(ROSA)26Sor-tdTomato reporter Ai14 mice (see Stock No. 007914) to create the triple mutation line. The Gad1 and Gad2 mutations are presumed to have crossed onto the same Chromosome 2 as mice can be made homozygous for both alleles. Triple homozygotes are viable and fertile. This strain was backcrossed to C57BL/6J for more than 6 generations by the donating laboratory.

Allele

Symbol: Gad1^tm1Rpa
Name: targeted mutation 1, Richard D Palmiter
MGI accession ID: MGI:3527168
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: Gad1^lox
Marker symbol: Gad1
Marker name: glutamate decarboxylase 1
Chromosome: 2
Strain of origin: Not Specified
Molecular note: Exon 2 was flanked by loxP site and a frt-flanked neo cassette was inserted downstream of exon 2. The neo cassette was removed by Flp-mediated recombination.

Allele

Symbol: Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}
Name: targeted mutation 14, Hongkui Zeng
MGI accession ID: MGI:3809524
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Rosa-CAG-LSL-tdTomato-WPRE::deltaNeo; Ai14; R26^Tom; STOP-RFP; R26R^T; Ai4; Rosa26-tdTomato; Rosa-lsl-tdTomato; Gt(ROSA)26Sor^{tm14(CAT-tdTomato)Hze}; Rosa26^Tdt; Rosa-LSL.dTm; Tomato^flox; tdTomato; R26-RFP; Gt(ROSA)26Sor^{tm9.1(CAG-tdTomato)Hze}; Gt(ROSA)26Sor_tm14(CAG/LSL_tdTomato)Hze; RCL-tdT
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: A loxP-flanked STOP cassette is designed to prevent transcription of the red fluorescent protein variant tdTomato. Upon exposure to Cre recombinase, robust tdTomato fluorescence may be observed. Importantly, the donating investigator reports that very low levels of tdTomato expression may be present prior to introduction of Cre recombinase - but the tdTomato expression levels after Cre recombination are significantly greater than those baseline levels.
Molecular note: The Rosa-CAG-LSL-tdTomato-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), tdTomato sequence (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. phiC31-recombination of Gt(ROSA)26Sortm9(CAG-tdTomato)Hze removed the neomycin resistance cassette.

Allele

Symbol: Gad2^tm1Rpa
Name: targeted mutation 1, Richard D Palmiter
MGI accession ID: MGI:6156077
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Synonyms: Gad2^lox
Marker symbol: Gad2
Marker name: glutamic acid decarboxylase 2
Chromosome: 2
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: Exon 1 was flanked by loxP site and a frt-flanked neo cassette was inserted downstream of exon 1.