C57BL/6N-Tg(Pvalb-tdTomato/RNAi:Gad1)3Mirn/J

Stock number: 028594
Product name: Pvalb-tdTomato-miGAD67
Strain synonyms: Pvalb/Gad1
Research areas: Cell Biology Research, Neurobiology Research, Research Tools, Sensorineural Research

Description

The Pvalb-tdTomato-miGAD67 line 3 BAC transgenic mouse line is a hybrid reporter/miRNA knockdown model where parvalbumin 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 PVALB+ GABAergic interneurons in behavioral and molecular processes, as well as cell-type specific GABAergic disturbances in manifestations of schizophrenia.

Detailed description

Pvalb-tdTomato-miGAD67 line 3 BAC transgenic mice have expression of both the tdTomato 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 parvalbumin 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 Pvalb gene; including fast-spiking inhibitory GABAergic interneurons of the cortex (basket cells and chandelier cells), and basket cells of the hippocampus, striatum and amygdala. PVALB+ cells exhibit non-detectable levels of GAD1 expression; resulting in reduced GABAergic synaptic transmission in layer V PL-PFC, altered sensorimotor gating deficits, increased novelty-seeking, diminished fear extinction and divergent dose-dependent NMDA antagonist (ketamine) responses. While fluorescence is faint, tdTomato expression via immunohistochemistry is reported specifically in PVALB+ cells (in hippocampus, dentate gyrus and neocortex).

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 tdTomato expression in PVALB+ cells.

Development

The Pvalb-tdTomato-miGAD67 line 3 BAC transgenic mouse line (also called Pvalb/Gad1) was created by Dr. Karoly Mirnics (Vanderbilt University). First, the 129.7 kbp bacterial artificial chromosome (BAC) RP24-306A6 was obtained possessing the parvalbumin locus (Pvalb) as well as ~65 kbp of centromeric sequences (including the complete Ift27 locus) and ~50 kbp of telomeric sequences (including the complete Ncf4 locus). Using homologous recombination/BAC recombineering, the following was inserted in-frame with the start codon of the BAC-encoded Pvalb: the red fluorescent protein tdTomato coding sequence, 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.

Also in vitro, the sequences encompassing exons 2-4 of the Ift27 locus were deleted. No other loci on the BAC were altered.

The ~140 kbp modified BAC was purified and microinjected into fertilized C57BL/6NTac mouse oocytes. Founder males were bred to C57BL/6NHsd females animals to establish germline transgenic lines. Founder line 3 was selected. The colony was backcrossed to C57BL/6NHsd for three 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(Pvalb-tdTomato/RNAi:Gad1)3Mirn
Name: transgene insertion 3, Karoly Mirnics
MGI accession ID: MGI:5703933
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); Reporter; Knockdown
Synonyms: Pvalb/Gad1
Marker symbol: Tg(Pvalb-tdTomato/RNAi:Gad1)3Mirn
Marker name: transgene insertion 3, Karoly Mirnics
Chromosome: UN
Strain of origin: C57BL/6NTac
Expressed genes: Gad1,glutamate decarboxylase 1,mouse, laboratory; RFP,Red Fluorescent Protein,coral
Site of expression: This transgene is expressed in the cell sub-populations that normally express the Pvalb gene; including fast-spiking inhibitory GABAergic interneurons of the cortex, and basket cells of the hippocampus, striatum and amygdala.
Molecular note: The transgenic construct uses 129.7 kbp from the bacterial artificial chromosome (BAC) RP24-306A6 possessing the parvalbumin locus (Pvalb) as well as ~65 kbp of centromeric sequences (including the complete Ift27 locus) and ~50 kbp of telomeric sequences (including the complete Ncf4 locus). Using homologous recombination/BAC recombineering, the following was inserted in-frame with the start codon of the BAC-encoded Pvalb: the red fluorescent protein tdTomato coding sequence, 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. Also in vitro, the sequences encompassing exons 2-4 of the Ift27 locus were deleted. No other loci on the BAC were altered.