C57BL/6-Cd74^em1Hrtn/PclbrJ

Stock number: 039411
Product name: Cd74^tdT
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

Cd74^tdT knock-in mice contain a CRISPR/Cas9-generated insertion of P2A-tdTomato-WPRE-pA replacing the endogenous stop codon of the Cd74 gene. These mice may be useful when monitoring MHC activation in diverse cell types and when studying their responses to inflammatory demyelination due to aging, injury, and disease.

Detailed description

Cd74^tdT knock-in mice contain a CRISPR/Cas9-generated insertion of P2A-tdTomato-WPRE-pA replacing the stop codon of the CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) (Cd74) gene. The presence of P2A allows for expression of both Cd74 and tdTomato (tdT) in these mice. The Cd74 gene encodes an MHC class II antigen which has diverse roles in cell survival, migration and MIF (macrophage migration inhibitory factor) cytokine signaling and is a required component of the antigen processing/presentation machinery expressed by a subset of oligodendroglia in mouse inflammatory models. Increased production of MHC in oligodendroglia of mice with multiple sclerosis (MS)-like symptoms is evident when compared with healthy animals, and the proportion of oligodendroglia that produce MHC is highest in mice with the most severe symptoms. MHC-expressing oligodendroglia have also been shown to congregate in the most damaged areas of the brain and spinal cord.

In these B2m^tdT mice, tdTomato fluorescence is highest in professional antigen presenting cells, such as dendritic cells and B cells. tdT expression was prevalent in the meninges and choroid plexus, with some expression evident in microglia in the ventral brain.

tdT fluorescent cells are immunoreactive to CD74 and are detectable by direct microscopy, flow cytometry, and immunofluorescence.

Heterozygous mice are viable and fertile. The donating investigator did not make a homozygous colony.

Development

A gRNA was designed to replace the stop codon of the endogenous CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) (Cd74) gene with a P2A self-cleaving peptide sequence, a red fluorescent protein (tdTomato) sequence, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) followed by a polyadenylation signal. Guide RNA targeting the stop codon (based on Cd74-202 ENSMUST00000097563.9), dsDNA donor plasmid (P2A-tdTomato-WPRE-pA) with 500bp homology arms, and the cas9 nuclease were electroporated into the cytoplasm of C57BL/6 derived embryos with well recognized pronuclei. Targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by PCR and fluorescence, and resulting Cd74^tdT mice were bred to C57BL/6J mice for at least 8 generations to remove off-target CRISPR sites by the donating laboratory. Upon arrival at The Jackson Laboratory, 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: Cd74^em1Hrtn
Name: endonuclease-mediated mutation 1, Cole Harrington
MGI accession ID: MGI:7666006
Generation method: Endonuclease-mediated
Attributes: Reporter
Synonyms: Cd74^tdT
Marker symbol: Cd74
Marker name: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated)
Marker synonyms: CLIP; DHLAG; HLADG; Ia-GAMMA; II; INVG34; p33
Chromosome: 18
Strain of origin: C57BL/6
Molecular note: Using CRISPR/Cas9 technology, a P2A self-cleaving peptide sequence, a red fluorescent protein (tdTomato) sequence, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) followed by a polyadenylation signal were inserted to replace the stop codon of the endogenous CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) (Cd74) gene.