STOCK Prokr2^em1(icre)Cfe/Mmjax

Stock number: 031786
Product name: ProkR2-Cre
Strain synonyms: PR2-Cre
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

ProkR2-Cre knock-in mice are designed to have codon optimized iCre recombinase expression directed to prokineticin receptor 2-expressing cells. These mice may be used to generate conditional mutations for studying gonadotropin-releasing hormone neuronal migration, olfactory bulb development, infertility, and Kallmann syndrome.

Detailed description

Prokr2 (prokineticin receptor 2) encodes a G protein-coupled receptor for prokineticin, a secreted chemokine. Prokr2 is mainly expressed in the central nervous system. Null mutations in Prokr2 and Prokr1 are associated with Kallmann syndrome, a form of hypogonadotropic hypogonadism that is characterized by lack of gonadotropin-releasing hormone neuronal migration (causing infertility) and impaired olfactory development resulting in a reduced or absent sense of smell. The ProkR2-Cre knock-in mutation has a T2A sequence and codon optimized iCre recombinase gene inserted immediately upstream of the Prokr2 termination codon - this is designed to have the endogenous promoter/enhancer elements direct iCre expression to PROKR2+ cells. Endogenous Prokr2 expression, gonadotropin-releasing hormone neuronal migration and fertility are not affected in these mice. ProkR2-Cre expression has a sexually dimorphic distribution. In females, expression is found in the medial preoptic area, ventrome-dial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral prabrachial nucleus. In males, expression occurs in the amygdalo-hippocampal area.

When ProkR2-Cre mice are bred with mice containing lox-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Prokr2-expressing cells in the offspring. These mice may be used to generate conditional mutations for studying gonadotropin-releasing hormone neuronal migration, olfactory bulb development, infertility, and Kallmann syndrome.

Development

This icre knock-in allele was created using homologous recombination (aided by targeted CRISPR/cas9 endonuclease activity) to insert a GSG linker, viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), a codon optimized Cre recombinase gene (iCre), and a polyA signal downstream of the endogenous termination codon of the prokineticin receptor 2 locus (Prokr2) on chromosome 2. The Cas9, the donor plasmid and the plasmid containing sgRNA were mixed and injected into the pronuclei of (C57BL/6 x SJL)F2 fertilized zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6J females for germline transmission. The colony was crossed to B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J or B6;129-Gt(ROSA)26Sor^tm2Sho/J and then maintained as a homozygous mating, reporter alleles were later bred out. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Prokr2^em1(icre)Cfe
Name: endonuclease-mediated mutation 1, Carol F Elias
MGI accession ID: MGI:6119460
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Synonyms: Prokr2-Cre
Marker symbol: Prokr2
Marker name: prokineticin receptor 2
Marker synonyms: B830005M06Rik; dJ680N4.3; EG-VEGRF2; Gpcr73l1; GPR73b; Gpr73l1; GPRg2; HH3; KAL3; PKR2
Chromosome: 2
Strain of origin: (C57BL/6 x SJL/J)F2
Expressed genes: cre,cre recombinase,bacteriophage P1; Prokr2,prokineticin receptor 2,mouse, laboratory
Site of expression: In females, expression of ProkR2-Cre is found in the medial preoptic area, ventrome-dial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral prabrachial nucleus. In males, expression occurs in the amygdalo-hippocampal area.
Molecular note: Using CRISPR/Cas9 technology, a GSG linker, a T2A viral peptide, an iCre recombinase, and a bovine growth hormone polyadenylation signal (bPA) were inserted between the final Prokr2 codon and termination codon. The sgRNA target was cloned into the plasmid pX330. The circular pX330 plasmidand circular donor plasmid were mixed and injected into the pronuclei of B6SJLF1 fertilized zygotes. Embryos were transferred to pseudopregnant B6D2F1 females to establish the mouse line.