Basic Information
-
Targeting strategy
-
Gene targeting strategy for B-hCCR8 mice(C). The full coding sequence and 3’UTR of mouse Ccr8 gene was replaced by human CCR8 counterpart gene in B-hCCR8 mice(C).
-
mRNA expression analysis
-
Strain specific analysis of CCR8 mRNA expression in wild-type BALB/c mice and B-hCCR8 mice(C) by RT-PCR. Thymus RNA was isolated from wild-type BALB/c mice (+/+), heterozygous (H/+) and homozygous B-hCCR8 mice(C) (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CCR8 primers. Mouse Ccr8 mRNA was detectable in wild-type BALB/c mice and heterozygous B-hCCR8 mice(C). Human CCR8 mRNA was detectable in heterozygous and homozygous B-hCCR8 mice(C) but not in wild-type mice.
-
CCR8 expression in spleen and thymus
-
Strain specific CCR8 expression analysis in B-hCCR8 mice(C) by flow cytometry. Splenocytes and thymocytes were collected from wild-type BALB/c mice (+/+), heterozygous (H/+) and homozygous (H/H) B-hCCR8 mice(C), and analyzed by flow cytometry with anti-CCR8 antibody. Mouse CCR8 was expression in T cells of thymocytes and expression lower in splenocytes that derived from wild-type and heterozygous mice, but not detectable in homozygous mice. Human CCR8 was expression lower in T cells of thymocytes and splenocytes that derived from all mice. Because the mouse CCR8 was not detectable in homozygous mice, the CCR8 expression in this mice was encoding from human CCR8 sequences. The anti-human CCR8 antibody may have species cross reaction between human and mouse.
-
hCCR8 (hCD198) expression in tumor infiltrating cells
-
CCR8 expression analysis in B-hCCR8 mice(C) by FACS. Colon cancer CT26.WT cells were inoculated into wild-type BALB/c (+/+) and homozygous B-hCCR8 mice(C) (H/H). Tumor cells were harvested after tumor growth and analyzed by flow cytometry. Human CCR8 was detectable in homozygous B-hCCR8 mice(C) as well as in wild-type BALB/c mice. The antibody may have species cross reaction between human and mouse.
-
mCCR8 (mCD198) expression in tumor infiltrating cells
-
CCR8 expression analysis in B-hCCR8 mice(C) by FACS. Colon cancer CT26.WT cells were inoculated into wild-type BALB/c (+/+) and homozygous B-hCCR8 mice(C) (H/H). Tumor cells were harvested after tumor growth and analyzed by flow cytometry. Mouse CCR8 was detectable in wild-type BALB/c mice and homozygous B-hCCR8 mice(C). The antibody may have species cross reaction between human and mouse.
-
hCCR8 (hCD198) expression in spleen
-
CCR8 expression analysis in B-hCCR8 mice(C) by FACS. Colon cancer CT26.WT cells were inoculated into wild-type BALB/c (+/+) and homozygous B-hCCR8 mice(C) (H/H). Spleen cells were harvested after tumor growth and analyzed by flow cytometry. Human CCR8 was not detectable in homozygous B-hCCR8 mice(C) and wild-type BALB/c mice.
-
mCCR8 (mCD198) expression in spleen
-
CCR8 expression analysis in B-hCCR8 mice(C) by FACS. Colon cancer CT26.WT cells were inoculated into wild-type BALB/c (+/+) and homozygous B-hCCR8 mice(C) (H/H). Spleen cells were harvested after tumor growth and analyzed by flow cytometry. Mouse CCR8 was not detectable in homozygous B-hCCR8 mice(C) and wild-type BALB/c mice.
-
Analysis of leukocytes cell subpopulation in blood
-
Analysis of blood leukocyte subpopulations by flow cytometry. Blood were isolated from female BALB/c and B-hCCR8 mice(C) (n=3, 7-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, DCs, granulocytes, monocytes and macrophages in homozygous B-hCCR8 mice(C) were similar to those in the BALB/c mice, demonstrating that CCR8 humanized does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
-
Summary
-
mRNA expression analysis
Human CCR8 mRNA was detectable in heterozygous and homozygous B-hCCR8 mice(C) but not in wild-type mice (page 6).Protein expression analysis
Human CCR8 was detectable in thymus of B-hCCR8 mice(C), but the protein level was lower than mouse CCR8 expression in wild-type mice (page 7).
Human CCR8 was expression lower (or not) in spleen of B-hCCR8 mice(C) (page 7, 10-11).
Human CCR8 was detectable in Tregs in TME that derived from homozygous B-hCCR8 mice(C) (page 8-9). Because the mouse CCR8 was not expressed anymore in homozygous mice according to the mRNA data, the CCR8 expression in this mice was encoding from human CCR8 sequences.leukocytes cell subpopulation analysis
Percent of T cells, B cells, NK cells, DCs, granulocytes, monocytes and macrophages in homozygous B-hCCR8 mice(C) were similar to those in the BALB/c mice, demonstrating that CCR8 humanized does not change the overall development, differentiation or distribution of these cell types in spleen, lymph node and blood.
