B6(Cg)-Brs3^em1(flpo)Rei/J

Stock number: 040970
Product name: Brs3-FlpO
Strain synonyms: Brs3-T2A-FlpO
Research areas: Neurobiology Research, Research Tools

Description

Brs3-FlpO knock-in mice are designed to express optimized FLP (Flpo) recombinase directed to bombesin-like receptor 3 (Brs3)-expressing cells. These mice may be used to generate conditional mutations for studying the function of Brs3-expressing cells, such as neurons involved in energy homeostasis and body temperature regulation.

Detailed description

Bombesin-like receptor 3, coded by Brs3, also known as Bombesin receptor subtype 3 (BB3), is a G protein-coupled receptor with functional roles in regulating energy metabolism, body temperature, and body weight. It is expressed in brain neurons that regulate feeding, body temperature, energy expenditure, and parenting behaviors. Brs3-FlpO knock-in (KI) mice harbor an optimized FLP recombinase (flpo) gene inserted at the Brs3 stop codon, while retaining about 80% of endogenous Brs3 RNA expression in PVH (paraventricular hypothalamus), as assayed by qPCR. This insertion is designed to direct flpo expression to Brs3-positive cells through the endogenous promoter/enhancer elements. When Brs3-FlpO KI mice are bred with mice containing frt-flanked sequences, FlpO-mediated recombination will result in deletion of the frt-flanked sequences in Brs3-expressing cells in the offspring.

The donating investigator reports FlpO recombinase expression/activity in Brs3-expressing cells in the medial preoptic area (MPOA), ventromedial preoptic area (VMPO), paraventricular nucleus of the hypothalamus (PVH), lateral hypothalamus (LH), dorsomedial hypothalamus (DMH), arcuate nucleus (ARC), and parabrachial nucleus (PBN) neurons among other brain regions where Brs3 expression has been detected. The donating investigator did not examine FlpO expression in tissues other than the brain. Homozygous females and hemizygous males are normal and fertile.

Development

The Brs3-FlpO knock-in allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. Guide RNAs (gRNA1: CAGTGTGCCAAGAAGGGAG-AGG, gRNA2: AACTTTGTCCTCTCCCTTCT-TGG) were used to introduce a 54-base pair (bp) T2A peptide followed by 1,299-bp codon-optimized flp-recombinase (FlpO) sequence into the stop codon of the bombesin-like receptor 3 (Brs3) gene on the X chromosome. Two synonymous mutations (Ala392, GCC to GCA; Glu396, GAG to GAA) were also introduced for CRISPR. Brs3 transcript Brs3-201 (NM_001727.2, ENST00000370648.4) was used as a reference for exon number and guide sequences. The sgRNAs, ssDNA, and the Cas9 nuclease were introduced into the cytoplasm of zygotes derived from C57BL/6 mice with well-recognized pronuclei. Correctly targeted pups were identified by PCR and sequencing. From the resulting Brs3-FlpO knock-in mice, one female founder (#1) was bred to C57BL/6J mice, and the other founder females (#2 and #3) were bred with FlpO reporter mice (RC::FLTG) B6.Cg-Gt(ROSA)26Sor^{tm1.3(CAG-tdTomato,-EGFP)Pjen}/J (Stock No. 026932). Female offspring from founder #1 and hemizygous Brs3-FlpO male without FlpO reporter from founder #2 and #3 were used to generate homozygous Brs3-FlpO females and hemizygous Brs3-FlpO males by the donating laboratory before sending hemizygous Brs3-FlpO males to the Jackson Laboratory. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Brs3^em1(flpo)Rei
Name: endonuclease-mediated mutation 1, Marc L Reitman
MGI accession ID: MGI:8234708
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Synonyms: Brs3-FlpO; Brs3-T2A-FlpO
Marker symbol: Brs3
Marker name: bombesin-like receptor 3
Marker synonyms: BB3; BB3R; BBR3; BRS-3
Chromosome: X
Strain of origin: C57BL/6
Molecular note: With a Gly-Ser-Gly (GGAAGCGGA) linker, 54-base pair (bp) T2A sequence and 1,299-bp FlpO sequence were inserted into the Brs3 stop codon using CRISPR technology. Two synonymous mutations (Ala392, GCC to GCA; Glu396, GAG to GAA) were also introduced for CRISPR.