C57BL/6-Cx3cr1^em1Mbur/J

Stock number: 037016
Product name: Cx3cr1^D2
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

Cx3cr1^D2 knock-in/knock-out mice express Dendra2 green/red photoswitchable monomeric fluorescent protein in myeloid cells including microglia and monocytes. Exposure of Dendra2-expressing cells to 405 nm laser light irreversibly switches green fluorescence to red, enabling labeling and tracking of cells.

Detailed description

Cx3cr1^D2 knock-in/knock-out mice express Dendra2 under direction of the endogenous Cx3cr1 promoter/enhancer elements, primarily in myeloid cells including microglia and monocytes. Dendra2 is a green-to-red, irreversibly photoswitchable monomeric fluorescent protein derived from octocoral Dendronephthya sp. In its unconverted state, Dendra2 has a peak excitation at 490 nm and a peak emission at 507 nm; however, when Dendra2-expressing cells are exposed to 405 nm laser light a structural photoconversion shifts the spectral properties to longer wavelengths, with a peak excitation of 553 nm and a peak emission of 573 nm enabling tracking of cells across time.

The donating investigator reports that immunohistochemical staining of Iba1 protein in fixed retinas of homozygous Cx3cr1^D2/D2 mice showed that Dendra2 was expressed in a large proportion of retinal microglia, as indicated by co-expression of Iba1. Green fluorescence prior in naive tissues, and red-orange fluorescence after photoactiviation by 405 nm laser light, is visible by microscopy without the aid of immunohistochemical amplification. Moreover, following photoconversion and subsequent damage of nearby photoreceptors using a high intensity near-infrared laser, the response of retinal microglia and the infiltration of monocytes to the injury site can be tracked over a period of at least 3 days by expression of the red-converted Dendra2 fluorophore (Miller et al., 2021).

Mice heterozygous or homozygous for the Cx3cr1^D2 allele are viable and fertile with no reported gross physical or behavioral abnormalities.

Development

The Cx3cr1-Dendra2 knock-in/knock-out allele (also called Cx3cr1^D2) was created using CRISPR/Cas9 endonuclease-mediated genome editing. Guide RNAs were selected to target exon 2 of the chemokine (C-X3-C motif) receptor 1 gene (Cx3cr1) on chromosome 9. Double stranded oligonucleotide donor DNAs were created to replace the first 130 amino acids of the endogenous Cx3cr1 locus with Dendra2 (a green to red irreversibly photoswitchable monomeric fluorescent protein derived from octocoral Dendronephthya sp.), an SV40 nuclear localization signal, and a polyadenylation sequence. These sequences and Cas9 mRNA were introduced into single cell C57BL/6N zygotes and transferred to pseudopregnant females. Progeny were screened by DNA sequencing to identify correctly targeted pups, which were then bred to C57BL/6J mice (Stock No. 000664) for germline transmission. The resulting Cx3cr1^D2 colony was then backcrossed to C57BL/6J mice for at least 1 generation prior to sending males to The Jackson Laboratory Repository in 2022. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Of note, the donating investigator reports that these mice have been screened for the absence of the Pde6b^rd1 and Crb1^rd8 mutations. In support of this, SNP testing of the mice sent to The Jackson Laboratory Repository were found to have wildtype SNPs in those two loci.

Allele

Symbol: Cx3cr1^em1Mbur
Name: endonuclease-mediated mutation 1, Marie Burns
MGI accession ID: MGI:6840220
Generation method: Endonuclease-mediated
Attributes: Reporter; Null/Knockout
Synonyms: Cx3cr1^D2; Cx3cr1-Dendra2
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: C57BL/6N
Molecular note: CRISPR/cas9 genome editing is used to target two sites in exon 2. Double stranded oligonucleotide donor DNAs are created to replace the first 130 amino acids of the endogenous Cx3cr1 locus with Dendra2 (a green to red irreversibly photoswitchable monomeric fluorescent protein derived from octocoral Dendronephthya sp.), an SV40 nuclear localization signal, and a polyadenylation sequence.