Overview

Also Known As:Gad1^lox::Gad2^lox::Rosa26^tdTomato

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.

Donating Investigator

Dr. Richard Palmiter, University of Washington

Genetic overview

Genetic Background	Generation

Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), Reporter)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

Gad1^tm1Rpa

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), No functional change)	Gad1	glutamate decarboxylase 1

Gad2^tm1Rpa

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed))	Gad2	glutamic acid decarboxylase 2

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Research Applications

Research Tools
Neurobiology Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

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.

Expression Data

Expressed Gene	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.
Site of Expression
Site of Expression

Control Suggestions

Approximate Controls

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

Meng F; Han Y; Srisai D; Belakhov V; Farias M; Xu Y; Palmiter RD; Baasov T; Wu Q. 2016. New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism. Proc Natl Acad Sci U S A 113(13):3645-50PubMed: 26976589 MGI: J:232044

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Genetics

Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}

Allele Symbol: Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}

Allele Name	targeted mutation 14, Hongkui Zeng
Allele Type	Targeted (Conditional ready (e.g. floxed), Reporter)
Allele Synonym(s)	Ai14; Ai4; Gt(ROSA)26Sor_tm14(CAG/LSL_tdTomato)Hze; Gt(ROSA)26Sor^{tm14(CAT-tdTomato)Hze}; Gt(ROSA)26Sor^{tm9.1(CAG-tdTomato)Hze}; R26^Tom; R26R^T; R26-RFP; RCL-tdT; Rosa26^Tdt; Rosa26-tdTomato; Rosa-CAG-LSL-tdTomato-WPRE::deltaNeo; Rosa-LSL.dTm; Rosa-lsl-tdTomato; STOP-RFP; tdTomato; Tomato^flox
Gene Symbol and Name	Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano
Gene Synonym(s)
Expressed Gene	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.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	6
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.
Mutations Made By	Hongkui Zeng, Allen Institute for Brain Science

Gad1^tm1Rpa

Allele Symbol: Gad1^tm1Rpa

Allele Name	targeted mutation 1, Richard D Palmiter
Allele Type	Targeted (Conditional ready (e.g. floxed), No functional change)
Allele Synonym(s)	Gad1^lox
Gene Symbol and Name	Gad1, glutamate decarboxylase 1
Gene Synonym(s)
Strain of Origin	Not Specified
Chromosome	2
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.

Gad2^tm1Rpa

Allele Symbol: Gad2^tm1Rpa

Allele Name	targeted mutation 1, Richard D Palmiter
Allele Type	Targeted (Conditional ready (e.g. floxed))
Allele Synonym(s)	Gad2^lox
Gene Symbol and Name	Gad2, glutamic acid decarboxylase 2
Gene Synonym(s)
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	2
Molecular Note	Exon 1 was flanked by loxP site and a frt-flanked neo cassette was inserted downstream of exon 1.

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Research Tools
- Fluorescent Proteins
- Cre-lox System
  - loxP-flanked Sequences
- Genetics Research
  - Tissue/Cell Markers: neurons
  - Mutagenesis and Transgenesis: Cre-lox System
  - Mutagenesis and Transgenesis: Production of Targeted Mutations (Knockouts)
  - Tissue/Cell Markers
- Neurobiology Research
  - cell marker
Neurobiology Research
- Neurotransmitter Receptor and Synaptic Vesicle Defects
- Cre-lox System
  - loxP-flanked Sequences
- Fluorescent protein expression in neural tissue

Mammalian Phenotype Terms by Genotype

References

Meng F; Han Y; Srisai D; Belakhov V; Farias M; Xu Y; Palmiter RD; Baasov T; Wu Q. 2016. New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism. Proc Natl Acad Sci U S A 113(13):3645-50PubMed: 26976589 MGI: J:232044

Additional - Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze} related

Additional - Gad1^tm1Rpa related

Additional - Gad2^tm1Rpa related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Separated MCA:Gt(ROSA)26Sor
- Standard PCR:Gt(ROSA)26Sor
- Standard PCR:Gt(ROSA)26Sor(tdTomato-WPRE)
- Standard PCR:Gad1
- Standard PCR:Gad2 Neo In
- Genotyping resources and troubleshooting
Breeding Considerations

Triple homozygous mutant mice are viable and fertile. The Gad1 and Gad2 mutations are presumed to have crossed onto the same Chromosome 2 as mice can be made homozygous for both alleles.
- Additional Breeding and Husbandry Support
Citation
When using the Gad1^lox::Gad2^lox::Rosa26^tdTomato mouse strain in a publication, please cite the originating article(s) and include JAX stock #031800 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service/Product	Description	Price
	Heterozygous for Gad2<tm1Rpa> , Gad1<tm1Rpa> and Gt(ROSA)26Sor<tm14(CAG-tdTomato)Hze>

Related Products and Services

Frozen Mouse Embryo	B6.Cg-Gad2<tm1Rpa> Gad1<tm1Rpa> Gt(ROSA)26Sor<tm14(CAG-tdTom	$2595.00
Frozen Mouse Embryo	B6.Cg-Gad2<tm1Rpa> Gad1<tm1Rpa> Gt(ROSA)26Sor<tm14(CAG-tdTom	$2595.00
Frozen Mouse Embryo	B6.Cg-Gad2<tm1Rpa> Gad1<tm1Rpa> Gt(ROSA)26Sor<tm14(CAG-tdTom	$3373.50
Frozen Mouse Embryo	B6.Cg-Gad2<tm1Rpa> Gad1<tm1Rpa> Gt(ROSA)26Sor<tm14(CAG-tdTom	$3373.50

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The Jackson Laboratory's Genotype Promise

The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.

Terms Of Use

General Terms and Conditions

Questions about Terms of Use

Additional Use Restrictions Apply

Use of MICE by companies or for-profit entities requires a license prior to shipping.

Licensing Information

Phone: 207-288-6470

Email: TechTran@jax.org

Also Known As:Gad1lox::Gad2lox::Rosa26tdTomato

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Gt(ROSA)26Sortm14(CAG-tdTomato)Hze

Allele Symbol: Gt(ROSA)26Sortm14(CAG-tdTomato)Hze

Gad1tm1Rpa

Allele Symbol: Gad1tm1Rpa

Gad2tm1Rpa

Allele Symbol: Gad2tm1Rpa

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

References

Additional - Gt(ROSA)26Sortm14(CAG-tdTomato)Hze related

Additional - Gad1tm1Rpa related

Additional - Gad2tm1Rpa related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

Also Known As:Gad1^lox::Gad2^lox::Rosa26^tdTomato

Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}

Allele Symbol: Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}

Gad1^tm1Rpa

Allele Symbol: Gad1^tm1Rpa

Gad2^tm1Rpa

Allele Symbol: Gad2^tm1Rpa

Additional - Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze} related

Additional - Gad1^tm1Rpa related

Additional - Gad2^tm1Rpa related