C57BL/6N-Tg(Cck-EGFP/RNAi:Gad1)2Mirn/J

Stock number: 028592
Product name: Cck-eGFP-miGAD67 line 2
Strain synonyms: Tg(Cck-eGFP/miRNA:GAD1)2KM , CCKGAD1TG
Research areas: Cell Biology Research, Neurobiology Research, Research Tools, Sensorineural Research

Description

The Cck-eGFP-miGAD67 line 2 BAC transgenic mouse line is a hybrid reporter/miRNA knockdown model where cholecystokinin 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 CCK+ GABAergic interneurons in behavioral and molecular processes, as well as cell-type specific GABAergic disturbances in manifestations of schizophrenia.

Detailed description

Cck-eGFP-miGAD67 line 2 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 cholecystokinin 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 Cck gene; including GABAergic interneuron basket cells (primarily synapse onto cell soma) or double bouquet cells (target dendrites in the amygdala, cortex, hippocampus, hypothalamus and thalamus). CCK+ cells exhibit non-detectable levels of GAD1 expression; resulting in decreased locomotor activity, increased olfactory investigation and reduced amphetamine sensitivity. While fluorescence is faint, strong eGFP expression via immunohistochemistry is reported specifically in CCK+ 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 CCK+ cells.

Development

The Cck-eGFP-miGAD67 line 2 BAC transgenic mouse line (also called Tg(Cck-eGFP/miRNA:GAD1)2KM or CCKGAD1TG) was created by Dr. Karoly Mirnics (Vanderbilt University). First, the 116 kbp bacterial artificial chromosome (BAC) RP23-60I1 was obtained possessing the cholecystokinin locus (Cck) as well as ~55 kbp of centromeric sequences (including a portion of the Trak1 locus) and ~56 kbp of telomeric sequences; no other confirmed complete 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 Cck: 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 ~127 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 2 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(Cck-EGFP/RNAi:Gad1)2Mirn
Name: transgene insertion 2, Karoly Mirnics
MGI accession ID: MGI:5703888
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); Reporter; Knockdown
Synonyms: CCKGAD1TG; Tg(Cck-eGFP/miRNA:GAD1)2KM
Marker symbol: Tg(Cck-EGFP/RNAi:Gad1)2Mirn
Marker name: transgene insertion 2, Karoly Mirnics
Chromosome: UN
Strain of origin: C57BL/6NHsd
Expressed genes: GFP,Green Fluorescent Protein,; Gad1,glutamate decarboxylase 1,mouse, laboratory
Site of expression: Expression of labeled interneurons is detected in the cell sub-populations that normally express the Cck gene, including GABAergic interneuron basket cells or double bouquet cells.
Molecular note: The transgenic construct uses 116 kbp from the bacterial artificial chromosome (BAC) RP23-60I1 containing the cholecystokinin locus (Cck) as well as ~55 kbp of centromeric sequences (including a portion of the Trak1 locus) and ~56 kbp of telomeric sequences; no other confirmed complete 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 Cck: 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.