The Cnr1-eGFP-miGAD67 line 1 BAC transgenic mouse line is a hybrid reporter/miRNA knockdown model where cannabinoid receptor 1 interneuronal expression of a synthetic Gad1 miRNA precursor results in Gad1 transcript downregulation (silencing of GAD1 expression) via endogenous miRNA processing mechanisms. These mice may be useful in studying CNR1+ GABAergic interneurons in behavioral and molecular processes, as well as cell-type specific GABAergic disturbances in manifestations of schizophrenia.
Karoly Mirnics, Vanderbilt University
Genetic Background | Generation |
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Allele Type |
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Transgenic (Conditional ready (e.g. floxed), Reporter, Knockdown) |
Cnr1-eGFP-miGAD67 line 1 BAC transgenic mice have expression of both the eGFP fluorescent protein and a synthetic miRNA sequence (miRNA:Gad1 or miGAD67) that targets the mouse glutamic acid decarboxylase 1 (Gad1 or GAD67) mRNA for knockdown. Expression is directed by the mouse cannabinoid receptor 1 promoter/enhancer regions on the BAC.
The morphology and distribution of the labeled interneurons across the different brain structures show that the transgene is specifically expressed in the cell sub-populations that normally express the Cnr1 gene; including GABAergic interneuron basket cells (smaller subpopulation of CCK+ basket cells).
CNR1+ cells exhibit non-detectable levels of GAD1 expression; resulting in complex behavioral and molecular disturbances, altered responses to cannabinoid and psychostimulant drugs, and increased preference for social novelty.
Strong eGFP fluorescence is reported specifically in CNR1+ cells across all tested brain regions (cortex, hippocampus, substantia nigra, amygdala and cerebellum), with strong labeling of neuronal somata and synaptic terminals.
Hemizygous mice are viable and fertile. To date (December 2015), it has not been attempted to make this strain homozygous.
The miRNA:Gad1-containing β-globin minigene is flanked with lox2272 sites, which allows excision of the GAD1 gene-silencing aspect of the transgene while retaining the eGFP fluorescence in CNR1+ cells.
The Cnr1-eGFP-miGAD67 line 1 BAC transgenic mouse line (also called Tg(Cnr1-eGFP/miRNA:Gad1) or Cnr1/Gad1) was created by Dr. Karoly Mirnics (Vanderbilt University). First, the 151.7 kbp bacterial artificial chromosome (BAC) RP24-370M5 was obtained possessing the cannabinoid receptor 1 locus (Cnr1) as well as ~86 kbp of centromeric sequences and ~41 kbp of telomeric sequences; no other confirmed genes are present in the flanking sequences (December 2015).
Using homologous recombination/BAC recombineering, the following was inserted in-frame with the start codon of the BAC-encoded Cnr1: the Enhanced Green Fluorescent Protein coding sequence (eGFP), a lox2272 site, a human β-globin minigene (exon1-intron-exon2) with the intron containing a miRNA:Gad1 sequence, a second lox2272 site and an SV40 polyA signal. A frt-flanked neomycin resistance cassette was also present, but removed during BAC recombineering in vitro.
The miRNA:Gad1 sequence is a fused miR30,miRNA:Gad1 synthetic miRNA sequence directed against the mouse glutamic acid decarboxylase 1 (Gad1 or GAD67) mRNA (mp ID 283874), with unique flanking miR-30 sequences.
No other loci on the BAC were altered.
The ~164 kbp modified BAC was purified and microinjected into fertilized C57BL/6NTac mouse oocytes. Founder males were bred to C57BL/6NHsd females to establish germline transgenic lines. Founder line 1 was selected. The colony was backcrossed to C57BL/6NHsd for four generations and then sent to The Jackson Laboratory in 2012.
Upon arrival, sperm was cryopreserved. If generating a live colony, an aliquot of frozen sperm may be used to fertilize C57BL/6NJ oocytes (Stock No. 005304).
Expressed Gene | GFP, Green Fluorescent Protein, |
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Expressed Gene | Gad1, glutamate decarboxylase 1, mouse, laboratory |
Site of Expression | EGFP fluorescence is reported in CNR1+ cells across all tested brain regions (cortex, hippocampus, substantia nigra, amygdala and cerebellum), with strong labeling of neuronal somata and synaptic terminals. |
Allele Name | transgene insertion 1, Karoly Mirnics |
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Allele Type | Transgenic (Conditional ready (e.g. floxed), Reporter, Knockdown) |
Allele Synonym(s) | Cnr1/Gad1; Cnr1-eGFP-miRNA:Gad1 |
Gene Symbol and Name | Tg(Cnr1-EGFP/RNAi:Gad1)1Mirn, transgene insertion 1, Karoly Mirnics |
Gene Synonym(s) | |
Promoter | Cnr1, cannabinoid receptor 1 (brain), mouse, laboratory |
Expressed Gene | GFP, Green Fluorescent Protein, |
Expressed Gene | Gad1, glutamate decarboxylase 1, mouse, laboratory |
Site of Expression | EGFP fluorescence is reported in CNR1+ cells across all tested brain regions (cortex, hippocampus, substantia nigra, amygdala and cerebellum), with strong labeling of neuronal somata and synaptic terminals. |
Strain of Origin | C57BL/6NTac |
Chromosome | UN |
Molecular Note | The transgenic construct uses 151.7 kbp from the bacterial artificial chromosome (BAC) RP24-370M5 possessing the cannabinoid receptor 1 locus (Cnr1) as well as ~86 kbp of centromeric sequences and ~41 kbp of telomeric sequences; no other confirmed genes are present in the flanking sequences. Using homologous recombination/BAC recombineering, the following was inserted in-frame with the start codon of the BAC-encoded Cnr1: eGFP, a lox2272 site, a human -globin minigene (exon1-intron-exon2) with the intron containing a miRNA:Gad1 sequence, a second lox2272 site and an SV40 polyA signal. An FRT-flanked neomycin resistance cassette was also present, but removed during BAC recombineering in vitro. The miRNA:Gad1 sequence is a fused miR30,miRNA:Gad1 synthetic miRNA sequence directed against the mouse glutamic acid decarboxylase 1 (Gad1 or GAD67) mRNA (mp ID 283874), with unique flanking miR-30 sequences. No other loci on the BAC were altered. |
When maintaining a live colony, hemizygous mice may be bred together, to wildtype (noncarrier) mice from the colony, or to C57BL/6NJ inbred mice (Stock No. 005304). To date (December 2015), it has not been attempted to make this strain homozygous.
When using the Cnr1-eGFP-miGAD67 mouse strain in a publication, please cite the originating article(s) and include JAX stock #028593 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Hemizygous or Non carrier for Tg(Cnr1-EGFP/RNAi:Gad1)1Mirn |
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