CLEC4C-DTR transgenic mice: A tool mouse strain generated for murine pDC depletion

• Gene Information: Human CLEC4C (BDCA 2/CD303), located on chromosome 12p13.31, encodes a transmembrane C-type lectin receptor selectively expressed on pDCs. The mouse gene Clec4c is not a direct ortholog of human CLEC4C and there is no definitive homologous gene for human CLEC4C in mice. Therefore, a safe-harbor locus was chosen for targeted knock-in. In this mouse strain, the focus is using the human CLEC4C promoter to drive a reporter cassette which is inserted immediately upstream of the CLEC4C stop codon.
• Protein Information: In this mouse line, human CLEC4C protein is expressed in a pDC-restricted manner, as driven by its own promoter. The EGFP-DTR-Luc fusion is co-produced (via a linker or as a fusion) from the same transcript, inserted upstream of the CLEC4C stop codon. This labels pDCs with EGFP, renders them sensitive to diphtheria toxin (DT) via DTR, and allows bioluminescence tracking via luciferase. The human CLEC4C protein itself here is not the functional focus; it merely serves as the carrier for the reporter cassette.
• Functional Mechanism: Although the full human CLEC4C coding sequence is present, the primary purpose of this model is to enable specific and inducible depletion of murine pDCs, not to study CLEC4C function. Upon DT injection, DTR on pDCs mediates toxin internalization and triggers rapid apoptosis. EGFP facilitates ex vivo flow cytometric identification, while luciferase permits non-invasive in vivo imaging of pDC distribution, migration, and clearance. Thus, this strain offers a robust platform for investigating pDC roles in disease models.

CLEC4C-EGFP-DTR-Luciferase

• The full coding sequence of the whole human CLEC4C genomic sequences with a EGFP-DTR-Luc reporter cassette before the stop codon was inserted into mouse Hipp11 gene site in B-hCLEC4C-EGFP-DTR-Luc mice.

EGFP expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCLEC4C-EGFP-DTR-Luc mice (male, 9-week-old, n = 3). EGFP expression on various types of immune cells was analyzed by flow cytometry.

EGFP expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCLEC4C-EGFP-DTR-luc mice (male, 9-week-old, n = 3). EGFP expression on various types of immune cells was analyzed by flow cytometry.

EGFP expression analysis in spleen DCs. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCLEC4C-EGFP-DTR-luc mice (male, 9-week-old, n = 3). EGFP expression on various types of DCs was analyzed by flow cytometry.

EGFP expression analysis in bone marrow cells. Bone marrow cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCLEC4C-EGFP-DTR-Luc mice (male, 9-week-old, n = 3). EGFP expression on various types of immune cells was analyzed by flow cytometry.

EGFP expression analysis in bone marrow cells. Bone marrow cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCLEC4C-EGFP-DTR-Luc mice (male, 9-week-old, n = 3). EGFP expression on various types of immune cells was analyzed by flow cytometry.

EGFP expression analysis in bone marrow DCs. Bone marrow cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCLEC4C-EGFP-DTR-Luc mice (male, 9-week-old, n = 3). EGFP expression on various types of DCs was analyzed by flow cytometry.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and bone marrow cells were isolated from male C57BL/6JNifdc and B-hCLEC4C-EGFP-DTR-Luc mice (male, 9-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Frequencies of cDC1, cDC2 and pDC in spleen by flow cytometry. Splenocytes and bone marrow were isolated from wild-type C57BL/6JNfidc mice (+/+) and homozygous B-hCLEC4C-EGFP-DTR-Luc mice (H/H) (n=3, male, 9-week-old) injected with PBS or DT (25 ng/g body weight) for four consecutive days. Flow cytometry analysis of the DCs in splenocytes and bone marrow cells was performed to assess the frequencies of cDC1, cDC2 and pDC in total CD11c+ cells. The frequency of pDC decreased in homozygous mice injected with DT.