B6.129S6-Gpr139^{tm1.1(flpo)Tasic}/J

Stock number: 034304
Product name: Gpr139-IRES2-FlpO-WPRE
Research areas: Neurobiology Research, Research Tools

Description

Gpr139-IRES2-FlpO-WPRE knock-in mice are designed to have optimized FLP recombinase expression directed to G protein-coupled receptor 139-expressing cells. FlpO expression/activity is observed in many locations throughout brain, with especially strong expression in olfactory bulb, striatum, lateral septum, hippocampus, amygdala, hypothalamus, midbrain, and strong scattered expression in layer 6 of cortex with less frequent scattered expression in other layers. These mice may be used to generate conditional mutations for studying GPR139 function in motor control, locomotor activity, metabolism, dopaminergic mesolimbic pathways, addiction, Parkinson's disease and schizophrenia.

Detailed description

G protein-coupled receptor 139 (Gpr139) is an orphan GPCR expressed in brain regions associated with regulation of motor control and metabolism, as well as dopaminergic mesolimbic pathways. Potential roles include addiction, Parkinson's disease, schizophrenia and locomotor activity.

The Gpr139-IRES2-FlpO-WPRE knock-in mutation has an IRES2 sequence and an optimized FLP recombinase (FlpO) gene inserted immediately downstream of the Gpr139 STOP codon - this is designed to have the endogenous promoter/enhancer elements direct FlpO expression to GPR139+ cells. When Gpr139-IRES2-FlpO-WPRE mice are bred with mice containing frt-flanked sequences, FlpO-mediated recombination will result in deletion of the frt-flanked sequences in Gpr139-expressing cells in the offspring.

The donating investigator reports FlpO recombinase expression/activity largely recapitulates the endogenous Gpr139 gene. Specifically, they report expression in many locations throughout brain, with especially strong expression in olfactory bulb, striatum, lateral septum, hippocampus, amygdala, hypothalamus, midbrain, and strong scattered expression in layer 6 of cortex with less frequent scattered expression in other layers. To date (April 2021), they have observed FlpO expressing cells mainly in layer 6 corticothalamic (CT) and layer 2/3 intratelencephalic (IT) excitatory neurons in primary visual cortex (they have not yet profiled neurons for other brain regions to pinpoint cellular identity of the labeled cells). The donating investigator did not examine FlpO expression in tissues other than brain.

This knock-in allele is designed to avoid disrupting expression of the endogenous protein, although Gpr139 expression from the Gpr139-IRES2-FlpO-WPRE allele has not been evaluated to date (April 2021).

Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. It has not been attempted to make this strain homozygous to date (April 2021).

Development

The Gpr139-IRES2-FlpO-WPRE mice were created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have an IRES2 sequence and an optimized FLP recombinase (FlpO) gene inserted immediately following the STOP codon of the G protein-coupled receptor 139 gene (Gpr139) on chromosome 7. The development of this strain included Addgene Plasmid #181767 and #181768. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Gpr139 sequence within exon 2 up to and including the endogenous stop codon, an internal ribosome entry site 2 sequence (IRES2; allows translation initiation in the middle of an mRNA sequence), a FlpO recombinase gene (a codon-optimized FLPe gene modified for translation in mammalian cells and higher recombination efficiency in cells and in mice), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA sequence, an AttB site, a PGK/gb2 promoter-Neomycin resistance gene-PGK polyA cassette and an AttP site. This construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked sequences (PGK/gb2-Neo-pA) and replace it with the recombined AttB/AttP site (AttL). The resulting Gpr139-IRES2-FlpO-WPRE mice were bred with C57BL/6J for several generations (and the PhiC31 transgene was removed). In 2021, males from generation N8 (black coat color) were sent to The Jackson Laboratory. Upon arrival, males are used to cryopreserve sperm. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Gpr139^{tm1.1(flpo)Tasic}
Name: targeted mutation 1.1, Bosiljka Tasic
MGI accession ID: MGI:6514363
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Gpr139^{tm1.1(flpo)Hze}
Marker symbol: Gpr139
Marker name: G protein-coupled receptor 139
Marker synonyms: GPRg1; LOC209776; PGR3
Chromosome: 7
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting vector contained, from 5' to 3', a partial Gpr139 sequence within exon 2 up to and including the endogenous stop codon, an internal ribosome entry site 2 sequence (IRES2; allows translation initiation in the middle of an mRNA sequence), a FlpO recombinase gene (a codon-optimized FLPe gene-modified for translation in mammalian cells and higher recombination efficiency in cells and in mice), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA sequence, an AttB site, a PGK/gb2 promoter-Neomycin resistance gene-PGK polyA cassette and an AttP site. Chimeric mice were bred to PhiC31-expressing mice to remove the AttB/AttP-flanked sequences (PGK/gb2-Neo-pA) and replace it with the recombined AttB/AttP site (AttL).