The Npy-eGFP-miGAD67 line 1 BAC transgenic mouse line is a hybrid reporter/miRNA knockdown model where neuropeptide Y 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 NPY+ 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) |
Npy-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 neuropeptide Y 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 Npy gene; including interneuron neurogliaform cells (primarily signal through nonsynaptic volume transmission) and Martinotti cells (synapse onto dendrites in amygdala, cortex, hippocampus, hypothalamus and striatum).
NPY+ cells exhibit non-detectable levels of GAD1 expression; resulting in reduced anxiety, increased preference for social novelty, increased PEP19/PCP4, increased amphetamine sensitivity.
Strong eGFP fluorescence is reported specifically in NPY+ cells.
The donating investigator reports that hemizygous and homozygous mice are viable and fertile.
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 NPY+ cells.
The Npy-eGFP-miGAD67 line 1 BAC transgenic mouse line (also called Tg(Npy-eGFP/miRNA:Gad1)1KM, NPY1, NPYGAD1TG or Npy/Gad1) was created by Dr. Karoly Mirnics (Vanderbilt University). First, the 160 kbp bacterial artificial chromosome (BAC) RP24-386I9 was obtained possessing the neuropeptide Y locus (Npy) as well as ~72 kbp of centromeric sequences and ~81 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 Npy: 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 ~173 kbp modified BAC was purified and microinjected into fertilized C57BL/6NHsd 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 seven 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 | The transgene is specifically expressed in the cell sub-populations that normally express the Npy gene; including interneuron neurogliaform cells and Martinotti cells. |
Allele Name | transgene insertion 1, Karoly Mirnics |
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Allele Type | Transgenic (Conditional ready (e.g. floxed), Reporter, Knockdown) |
Allele Synonym(s) | NPRGAD1TG; NPY BAC/GAD1 miRNA; Tg(Npy-eGFP/miRNA:Gad1)1KM |
Gene Symbol and Name | Tg(Npy-EGFP/RNAi:Gad1)1Mirn, transgene insertion 1, Karoly Mirnics |
Gene Synonym(s) | |
Promoter | Npy, neuropeptide Y, mouse, laboratory |
Expressed Gene | GFP, Green Fluorescent Protein, |
Expressed Gene | Gad1, glutamate decarboxylase 1, mouse, laboratory |
Site of Expression | The transgene is specifically expressed in the cell sub-populations that normally express the Npy gene; including interneuron neurogliaform cells and Martinotti cells. |
Strain of Origin | C57BL/6NHsd |
Chromosome | UN |
Molecular Note | The transgenic construct uses 160 kbp from bacterial artificial chromosome (BAC) RP24-386I9 containing the neuropeptide Y locus (Npy) as well as ~72 kbp of centromeric sequences and ~81 kbp of telomeric sequences; no other confirmed genes are present in the flanking sequences. Using homologous recombination/BAC recombineering, the following is inserted in-frame with the start codon of the BAC-encoded Npy: 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. |
The donating investigator reports that hemizygous and homozygous mice are viable and fertile. 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). Alternatively, homozygous mice may be bred together.
When using the Npy-eGFP-miGAD67 line 1 mouse strain in a publication, please cite the originating article(s) and include JAX stock #028591 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Hemizygous or Non carrier for Tg(Npy-EGFP/RNAi:Gad1)1Mirn |
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