Basic Information
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Targeting strategy
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Gene targeting strategy for B-Cd11c EGFP-DTR-Luc mice.
CD11c co-express EGFP, the DTR for depletion of DCs, and the luciferase for in vivo bioluminescent imaging (BLI) of DCs. EGFP-DTR-Luc expression is evident in CD11c+ cells.
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Protein expression analysis-Spleen
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Strain EGFP expression analysis in heterozygous B-Cd11c-EGFP-DTR-Luc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd11c-EGFP-DTR-Luc mice (Mut/+), and analyzed by flow cytometry. EGFP was detectable in NK cells, DCs, monocytes and macrophages of heterozygous B-Cd11c-EGFP-DTR-Luc mice, but not in wild-type C57BL/6 mice.
Frequencies of EGFP+ cells and DCs in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd11c-EGFP-DTR-Luc mice (Mut/+) (n=3, 6-8-week-old) injected with PBS or DT (50 ng/g body weight) for two consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequencies of EGFP+ cells and DCs. The frequencies of EGFP+ cells and DCs were decreased in heterozygous mice injected with DT.
Frequencies of NK cells and neutrophils in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd11c-EGFP-DTR-Luc mice (Mut/+) (n=3, 6-8-week-old) injected with PBS or DT (50 ng/g body weight) for two consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequencies of NK cells and neutrophils. The frequencies of NK cells was decreased in heterozygous mice injected with DT, while the frequency of neutrophils was increased.
Frequencies of monocytes and macrophages in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd11c-EGFP-DTR-Luc mice (Mut/+) (n=3, 6-8-week-old) injected with PBS or DT (50 ng/g body weight) for two consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequencies of monocytes and macrophages. The frequency of monocytes was decreased in heterozygous mice injected with DT, while the frequency of macrophages was increased.
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Frequency of leukocyte subpopulations in spleen
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Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd11c-EGFP-DTR-Luc mice (Mut/+) (n=3, 6-8-week-old) injected with PBS or DT (50 ng/g body weight) for two consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***p < 0.0001.
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Summary
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Protein expression analysis:
- EGFP was only detectable in heterozygous B-Cd11c-EGFP-DTR-Luc mice, but not in wild-type C57BL/6 mice.
- EGFP was only detectable on DCs, NK cells, monocytes and macrophages from spleen of heterozygous B-Cd11c-EGFP-DTR-Luc mice.
Functional analysis:
- The frequencies of DCs and EGFP+ cells were decreased in heterozygous B-Cd11c-EGFP-DTR-Luc mice with injected with DT.
- The frequencies of NK cells and monocytes were decreased in heterozygous B-Cd11c-EGFP-DTR-Luc mice with injected with DT, while the frequencies of neutrophils and macrophages were increased.
