Description
- B-Cd8a-EGFP-DTR-luciferase mice have the endogenous mouse Cd8a promoter to determine the expression of DTR (Diphtheria toxin receptor), EGFP (enhanced green fluorescent protein) and luciferase.
- CD8+ T cells and cDC1 cells were traced by EGFP in B-Cd8a-EGFP-DTR-luciferase mice, and could be effectively eliminated after DT administration.
Targeting strategy
Gene targeting strategy for B-Cd8a-EGFP-DTR-luciferase mice. A construct composed of an internal ribosome entry site (IRES), the cDNA for enhanced GFP (EGFP), the human DTR, and luciferase was inserted at the stop codon of the Cd8a gene in B-Cd8a-EGFP-DTR-luciferase mice, allowing for EGFP, DTR and luciferase expression and de novo CD8a expression.
Total CD8+ cells depletion analysis
Frequencies of CD8a+ cells in spleen and blood by flow cytometry. Splenocytes and blood were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd8a-EGFP-DTR-luciferase mice (Mut/+) (n=3, 7-8-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes and blood was performed to assess the frequency of CD8a+; EGFP+ cells in total CD45+ cells. The signal for CD8a+; EGFP+ cells in spleen(A) and CD8a+; EGFP+ cells in blood (B) were significant decreased in heterozygous mice injected with DT.
Total CD8+ cells depletion analysis in spleen
Frequencies of CD8a+ cells and EGFP+ cells in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd8a-EGFP-DTR-luciferase mice (Mut/+) (n=3, 7-8-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequencies of CD8a+ cells and EGFP+ cells in total CD45+ cells. The signal for CD8a+ cells(A) and EGFP+ cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of CD8a+ cells and EGFP+ cells in total CD45+ populations. The frequencies of CD8a+ cells and EGFP+ cells were decreased in heterozygous mice after DT injection.
Total CD8+ cells depletion analysis in blood
Frequencies of CD8a+ cells and EGFP+ cells in blood by flow cytometry. Blood were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd8a-EGFP-DTR-luciferase mice (Mut/+) (n=3, 7-8-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the blood cells was performed to assess the frequencies of CD8a+ cells and EGFP+ cells in total CD45+ cells. The signal for CD8a+ cells(A) and EGFP+ cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of CD8a+ cells and EGFP+ cells in total CD45+ populations. The frequencies of CD8a+ cells and EGFP+ cells were decreased in heterozygous mice after DT injection.
CD8+ T cells depletion analysis
Frequencies of CD8+ T cells in spleen and blood by flow cytometry. Splenocytes and blood were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd8a-EGFP-DTR-luciferase mice (Mut/+) (n=3, 7-8-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes and blood was performed to assess the frequency of CD8+ T cells in total CD45+ cells. The signal for CD8+ T cells in spleen(A) and CD8+ T cells in blood (B) were significant decreased in heterozygous mice injected with DT. C. Percentages of CD8+ T cells in total CD45+ populations. The frequencies of CD8+ T cells in spleen and blood were decreased from heterozygous mice after DT injection.
Frequencies of EGFP in CD8+ T cells from spleen and blood by flow cytometry. Splenocytes and blood were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd8a-EGFP-DTR-luciferase mice (Mut/+) (n=3, 7-8-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes and blood was performed to assess the frequency of EGFP cells in total CD8+ T cells. The signal for EGFP in CD8+ T cells from spleen(A) and EGFP in CD8+ T cells from blood (B) were significant decreased in heterozygous mice injected with DT. C. Percentages of EGFP in CD8+ T cells from spleen and blood. The frequencies of EGFP in total CD8+ T cells were significant decreased both in spleen and blood from heterozygous mice after DT injection.
CD8a+ Dendritic cells(cDC1) depletion analysis
Frequency of EGFP in cDC1 cells from spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd8a-EGFP-DTR-luciferase mice (Mut/+) (n=3, 7-8-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequency of EGFP in total cDC1 cells. The signal for cDC1 cells(A) and EGFP in cDC1 cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of cDC1 cells in total DC populations. D. Percentages of EGFP in total cDC1 cells. The frequencies of cDC1 cells and EGFP in cDC1 cells were decreased in heterozygous mice after DT injection.
Frequency of leukocyte subpopulations in spleen
Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and B-Cd8a-EGFP-DTR-luc mice(n=3, 7-8-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, NK cells, dendritic cells, granulocytes, monocytes, macrophages and Tregs in heterozygous B-Cd8a-EGFP-DTR-luc mice were similar to those in C57BL/6 mice. While the percentage of CD8+ T cells showed significant difference between heterozygous B-Cd8a-EGFP-DTR-luc mice and wild-type mice. The frequency of leukocyte subpopulations in blood and lymph node of B-Cd8a-EGFP-DTR-luc mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM.
