Gene targeting strategy for B-hCLEC9A mice. The part of exon 2 and exon 3~6 of mouse Clec9a gene that encode transmembrane region and extracellular region were replaced by human counterparts in B-hCLEC9A mice. The genomic region of mouse Clec9a gene that encodes cytoplasmic portion was retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The CLEC9A expression was driven by endogenous mouse Clec9a promoter, while mouse Clec9a gene transcription and translation will be disrupted.

Strain specific CLEC9A expression analysis in homozygous B-hCELC9A mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCLEC9A mice (H/H), and analyzed by flow cytometry with species-specific anti-CLEC9A antibodies. Mouse CLEC9A was detectable in mCD8α+ DCs and mCD103+ DCs of wild-type mice. Human CLEC9A was exclusively detectable in mCD8α+ DCs and mCD103+ DCs of homozygous B-hCLEC9A mice but not in wild-type mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry.Splenocytes were isolated from wild-type C57BL/6 mice (female, n=3, 6-week-old) and homozygous B-hCLEC9A mice (female, n=3, 6-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCLEC9A mice were similar to those in C57BL/6N mice. Values are expressed as mean ± SEM.