B6.129P2(Cg)-Cx3cr1^tm1Litt/J

Stock number: 005582
Product name: CX₃CR-1^GFP
Research areas: Cancer Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

CX₃CR-1^GFP knock-in/knock-out mice express EGFP in monocytes, dendritic cells, NK cells, and brain microglia under control of the endogenous Cx3cr1 locus. They may be useful in studies of leukocyte migration and trafficking, as well as for transplantation studies.

Detailed description

CX₃CR-1^GFP knock-in/knock-out mice have an enhanced green fluorescent protein (EGFP) sequence replacing the first 390 bp of the coding exon (exon 2) of the chemokine (C-X3-C motif) receptor 1 (Cx3cr1) gene. RT-PCR analysis of lymphoid tissue from homozygotes detects mutant gene product (mRNA) and no wild type gene product (mRNA). Flow cytometric analysis of peripheral blood cells identified a subset of green fluorescent cells not observed in wildtype mice. EGFP, but not the endogenous gene, is expressed in monocytes, dendritic cells, NK cells, and brain microglia - mimicking endogenous gene expression. The same subset of peripheral blood cells isolated from heterozygote mice express detectable levels of EGFP. These CX₃CR-1^GFP mutant mice may be useful in studies of leukocyte migration and trafficking, as well as for transplantation studies.

Mice that are homozygous for the CX₃CR-1^GFP targeted mutation are viable and fertile.

Of note, CX₃CR-1^GFP mice are also available harboring with the CD45.1 (Ly5.1 or Ptprc^a) allele, which is atypical for the C57BL/6 congenic background (see Stock No. 008451).

When compared with GFP expression in monocytes in C57BL/6-Tg(CD68-EGFP)1Drg/J mice (Stock No. 026827) using a sterile zymosan peritonitis model, CD68-GFP monocytes retain high-level GFP expression as they differentiate to become mature macrophages, while, CX₃CR1^GFP monocytes downregulate GFP expression on differentiation into macrophages.

Development

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. The donating investigator reported that resulting chimeric animals were backcrossed to C57BL/6 (see SNP note below) for ten generations before being made homozygous. During the backcross, mice were likely bred to a B6.CD45.1 congenic strain (harboring the CD45.1 (Ly5.1 or Ptprc^a) allele rather than the CD45.2 (Ly5.2 or Ptprc^b) allele normally present in C57BL/6 mice). These mice, however, were bred such that they still harbor the expected CD45.2 (Ly5.2 or Ptprc^b) allele normally present in C57BL/6 mice.

In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Three of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Cx3cr1^tm1Litt
Name: targeted mutation 1, Dan R Littman
MGI accession ID: MGI:2670351
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Cx3cr1
Marker name: chemokine (C-X3-C motif) receptor 1
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 coding exon (exon 2). 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.