Basic Information

Strain Name
C57BL/6-Tnfsf11tm1(TNFSF11)/Bcgen
Stock Number
111072
Common Name
B-hRANKL mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Aliases
CD254, ODF, OPGL, OPTB2, RANKL, TNLG6B, TRANCE, hRANKL2, sOdf
Species
C57BL/6J
Appearance
Black
Genotypes
Homozygous
NCBI Gene ID

Gene targeting strategy

Gene targeting strategy for B-hRANKL mice. The exons 4~5 of mouse Rankl gene that encode the extracellular region were replaced with human RANKL exons 4~5 in B-hRANKL mice.

mRNA expression analysis

Species-specific RANKL gene expression analysis in wild-type and humanized B-hRANKL mice by RT-PCR. Murine Rankl mRNA was detected in thymocytes isolated from wild-type C57BL/6 (+/+) mice, while human RANKL mRNA was exclusively detected in homozygous B-hRANKL (H/H) mice.

Protein expression analysis

Species-specific RANKL protein expression analysis in wild-type and humanized B-hRANKL mice. Splenocyte-derived CD4+ T cells were isolated from wild-type C57BL/6 (+/+) and homozygous B-hRANKL (H/H) mice, stimulated with anti-mCD3e and anti-mCD28 antibodies in vitro, and analyzed by flow cytometry using species-specific anti-RANKL antibodies. Murine RANKL protein was detected in wild-type mice, while human RANKL protein was exclusively detected in B-hRANKL mice.

Immune cell profiling

Analysis of leukocytes cell subpopulation in spleen

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hRANKL mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes 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, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hRANKL mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hRANKL in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in spleen

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hRANKL mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hRANKL mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hRANKL in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in lymph node

Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hRANKL mice (n=3, 6-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 and NK cells in homozygous B-hRANKL mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hRANKL in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in lymph node

Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hRANKL mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hRANKL mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hRANKL in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in blood

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hRANKL mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells 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, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hRANKL mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hRANKL in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in blood

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hRANKL mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hRANKL mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hRANKL in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

In vivo efficacy of an anti-human RANKL Ab in an osteoporosis B-hRANKL mouse model

In vivo efficacy of an anti-human RANKL antibody denosumab (in house) in an osteoporosis B-hRANKL mouse model. Mice were randomly separated to receive ovariectomy or sham surgery. The ovariectomized mice were then randomly divided into 3 treatment groups (OVX, Denosumab and PTH1-34) 4 weeks after surgery (n = 6). Mice were sacrificed at 4 weeks after treatment. Serum was obtained 3 times before and after treatment for analysis of C-terminal telopeptide of type 1 collagen (CTX-1) and osteocalcin (OC). The time points of serum collection are shown in (A). The proximal tibia was harvested for analysis of bone mineral density (BMD) by μCT. A-B. The concentration of serum bone resorption marker CTX-1 and serum bone formation marker OC. C. The BMD change of the proximal tibia. Results showed that compared with the sham group without surgery, the concentration of bone resorption marker CTX-1 was significantly increased, while the concentration of bone formation marker OC and BMD change of proximal tibia were significantly decreased in the OVX group, indicating that the osteoporosis mouse model was successfully modeled with B-hRANKL mice. After treatment with anti-human RANKL antibody or PTH, the concentration of CTX-1 was significantly decreased, while the concentration of OC and the percentage change of BDM were significantly increased compared with the untreated OVX group. These results indicated that the anti-human RANKL antibody could effectively treat osteoporosis in B-hRANK mice. The B-hRANKL mice provide a powerful preclinical mouse model for in vivo evaluating efficacy of anti-human RANKL antibodies. Values are expressed as mean ± SEM.