C57BL/6N-Tg(Npy-EGFP/RNAi:Gad1)1Mirn/J

Stock number: 028591
Product name: Npy-eGFP-miGAD67 line 1
Strain synonyms: Tg(Npy-eGFP/miRNA:Gad1)1KM , NPY1 , NPYGAD1TG , Npy/Gad1
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

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.

Detailed description

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.

Development

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).

Allele

Symbol: Tg(Npy-EGFP/RNAi:Gad1)1Mirn
Name: transgene insertion 1, Karoly Mirnics
MGI accession ID: MGI:5703795
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); Reporter; Knockdown
Marker symbol: Tg(Npy-EGFP/RNAi:Gad1)1Mirn
Marker name: transgene insertion 1, Karoly Mirnics
Chromosome: UN
Strain of origin: C57BL/6NHsd
Expressed genes: GFP,Green Fluorescent Protein,; 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.
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.