B6.Cg-Ptprc^a Cx3cr1^tm1Litt/LittJ

Stock number: 008451
Product name: CX₃CR1^GFP
Research areas: Cancer Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

These CX3CR1-GFP mice express EGFP under control of the endogenous Cx3cr1 locus and also harbor the CD45.1 (Ly5.1 or Ptprc^a) allele, which is atypical for the C57BL/6 congenic background and may be useful in studies of leukocyte migration and trafficking, as well as for transplantation studies with C57BL/6 (CD45.2: Ptprc^b) mice.

Detailed description

Mice that are homozygous for the CX3CR1-GFP targeted mutation are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. 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 wild type mice. Enhanced Green Fluorescent Protein (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 mice also express the CD45.1 (Ly5.1 or Ptprc^a) allele, which is atypical for the C57BL/6 congenic background, and this marker may be used to track donor cell populations in transplantation studies with C57BL/6 (CD45.2, Ly5.2 or Ptprc^b) mice. These CX3CR1-GFP mice may be useful in studies of leukocyte migration and trafficking, as well as for transplantation studies.

Of note, CX3CR1-GFP mice are also available with the Ptprc^b allele normally found in the C57BL/6 genetic background (see Stock No. 005582).

Development

To generate the Cx3cr1 mutant 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. The resulting chimeric animals were backcrossed to C57BL/6 for ten generations before being made homozygous. During the backcross, mice were likely bred to a B6.CD45.1 congenic strain and thus also harbor 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. Upon arrival at The Jackson Laboratory Repository, mice were bred with C57BL/6J inbred mice to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating (one on chromosome 11 and one on chromosome 13). Breeding mutant mice together at The Jackson Laboratory Repository for several generations may have fixed these 2 markers as C57BL/6N allele-type.

Allele

Symbol: Ptprc^a
Name: a variant
MGI accession ID: MGI:4819849
Generation method: Not Applicable
Marker symbol: Ptprc
Marker name: protein tyrosine phosphatase, receptor type, C
Chromosome: 1
Strain of origin: Not Applicable
Site of expression: Widely expressed on all adaptive and innate immune cells.
Molecular note: Ptprc^a is found in strains SJL/J, STS/A, and DA. Ptprc^b is found in strains C57BL/6, C3H/An, DBA/2, AKR, and many others (J:13367, J:12054, J:12077, J:8603). Twelve nucleotide differences between the a and b alleles have been identified. These base substitutions correspond to five amino-acid changes within the extracellular domain of the encoded protein. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat sequences (J:22485).

Note that the allele designations originally described were reversed in 1987 (J:8603); all publications prior to 1987 show SJL/J, STS/A, and DA as having the b allele and the C57BL/6J group as having the a allele (J:22341).

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.