B6.129(Cg)-Cx3cr1^tm1Litt Ccr2^tm2.1Ifc/JernJ

Stock number: 032127
Product name: Ccr2^RFPCx3cr1^GFP
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

Ccr2^RFPCx3cr1^GFP dual-reporter mice express red fluorescent protein in CCR2 positive peripheral monocytes, T cells and NK cells; and green fluorescent protein in monocytes, dendritic cells, NK cells and brain microglia. Both Ccr2^RFP and Cx3cr1^GFP are knock-in/knock-out alleles. These mice may be suitable for use in studies tracking myeloid lineage cell subpopulations, monocyte recruitment in inflammatory responses, leukocyte migration and trafficking, as well as for transplantation studies.

Detailed description

CCR2 is a chemokine receptor expressed on monocytes which directs their recruitment to sites of inflammation found in diseases such as multiple sclerosis, atherosclerosis, rheumatoid arthritis, and experimental autoimmune encephalitis (EAE), following activation by monocyte chemoattractant proteins. The Cx3cr1 gene encodes the receptor for C-X3-C motif chemokine (CX3CR1), also known as fractalkine, which is involved in leukocyte adhesion and migration. The Cx3cr1 and Ccr2 genes are located in close proximity on mouse Chromosome 9.
These Ccr2^RFPCx3cr1^GFP dual-reporter mutant mice, also called R2X3-RGFP, carry a monomeric red fluorescent protein (RFP) knock-in/knock-out allele of Ccr2 and a green fluorescent protein (EGFP) knock-in/knock-out allele of Cx3cr1. Red fluorescent protein expression is detected in CCR2 positive peripheral monocytes, T cells and NK cells; and green fluorescent protein expression is detected in monocytes, dendritic cells, NK cells and brain microglia. Mice that are homozygous for both mutations are viable and fertile. These double homozygotes lack functional CCR2 and CX3CR1 receptors. Double homozygous mice exhibit impaired monocyte recruitment and enhanced susceptibility to experimental autoimmune encephalomyelitis.

Development

Ccr2^RFPCx3cr1^GFP dual-reporter mice (Stock No. 032127) is a double mutant strain produced by crossing B6.129(Cg)-Ccr2^tm2.1Ifc/J (Stock No. 017586) and B6.129P-Cx3cr1^tm1Litt/J (Stock No. 005582). Each mouse line is described in more detail below. Because the Cx3cr1 and Ccr2 genes are located in close proximity on mouse Chromosome 9, generation of the double homozygotes required extensive crosses.

For the Cx3cr1^tm1Litt allele, a targeting vector containing an Enhanced Green Fluorescent Protein (EGFP, Clontech) cDNA sequence, loxP-flanked neomycin resistance gene, herpes simplex virus thymidine kinase gene, and SV40 polyadenylation site sequence was used to disrupt the first 390 bp of exon 2. The construct was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells which were transiently transfected with a Cre recombinase vector to remove the selection cassette. ES cells that had successfully undergone Cre-mediated recombination (removing the loxP-flanked neo cassette and leaving a single loxP site downstream of EGFP) were injected into recipient blastocysts. Chimeras were bred to C57BL/6 for germline transmission. The colony was backcrossed approximately ten generations to C57BL/6 mice (which likely included a B6.CD45.1 congenic strain harboring the CD45.1 [Ly5.1 or Ptprc^a] allele). C57BL/6-congenic Cx3cr1^GFP mice (harboring the expected CD45.2 [Ly5.2 or Ptprc^b] allele normally present in C57BL/6 animals) were sent to The Jackson Laboratory as Stock No. 005582.

For the Ccr2^tm2.1Ifc allele, a targeting vector was designed to replace the coding sequence of the chemokine (C-C motif) receptor 2 (Ccr2) gene with a monomeric red fluorescent protein (RFP) sequence followed by a polyadenylation signal and a loxP-flanked neomycin resistance cassette. The construct was electroporated into 129-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric mice were bred to C57BL/6 mice. The resulting offspring were bred to Tg(EIIa-cre)C5379Lmgd transgenic mice to delete the neo cassette. C57BL/6-congenic Ccr2^RFP mice were sent to The Jackson Laboratory as Stock No. 017586.

 

Allele

Symbol: Ccr2^tm2.1Ifc
Name: targeted mutation 2.1, Israel F Charo
MGI accession ID: MGI:4868431
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: Ccr2^R; Ccr2^RFP
Marker symbol: Ccr2
Marker name: chemokine (C-C motif) receptor 2
Marker synonyms: CC-CKR-2; CCR-2; CCR2A; CCR2B; CD192; CKR2; CKR2A; CKR2B; CMKBR2; MCP-1-R; PCLUD
Chromosome: 9
Strain of origin: 129
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: The red fluorescent protein (RFP) reporter allows visualization of Ly6C^hi monocytes.
Molecular note: The coding region was replaced with an RFP and floxed neo cassette. Cre-mediated recombination removed the neo cassette. The absence of transcript expression was confirmed by RT-PCR.

Allele

Symbol: Cx3cr1^tm1Litt
Name: targeted mutation 1, Dan R Littman
MGI accession ID: MGI:2670351
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: CX3CR1^-; CX3CR1^EGFP; Cx3cr1^gfp; CX3CR1-EGFP; CX3CR1-GFP; CX3CR-GFP
Marker symbol: Cx3cr1
Marker name: chemokine (C-X3-C motif) receptor 1
Marker synonyms: CCRL1; CMKBRL1; CMKDR1; GPR13; GPRV28; Rbs11; V28
Chromosome: 9
Strain of origin: 129P2/OlaHsd
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: GFP labels T and B cells in the lymphoid organs such as spleen, small intestine, and large intestine.
Molecular note: The endogenous locus was disrupted by the insertion of sequence encoding green fluourescent protein (GFP), replacing the first 390 bp of the only coding exon. The deleted region encoded an amino-terminal portion of the protein that is crucial for interaction with endogenous ligand, Cx3cl1. A floxed neo gene included in the targeting vector for selection was excised prior to germline transmission, leaving a single loxP site downstream of the GFP sequence. RT-PCR and flow cytometry indicated an absence of endogenous protein and the presence GFP expression in homozygous mutant mice.