Basic Information
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Targeting strategy
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Gene targeting strategy for B-Cd19-EGFP-DTR-luciferase mice.
A construct composed of the cDNA for enhanced GFP (EGFP), the human DTR, and luciferase was inserted before the stop codon of the Cd19 gene in B-Cd19-EGFP-DTR-luciferase mice, allowing for EGFP, DTR and luciferase expression and de novo Cd19 expression. The coding sequences were separated by self-cleaving 2A peptide sequences.
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EGFP expression analysis in leukocytes cell subpopulation
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Frequencies of EGFP in leukocytes cell subpopulation from spleen, blood and lymph node by flow cytometry. Splenocytes, blood and lymph node were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old). Flow cytometry analysis of the splenocytes, blood and lymph node was performed to assess the frequencies of EGFP+ cells in leukocytes cell subpopulation from spleen(A), blood (B) and lymph node(C). EGFP+ cells was expressed at a high level in B cells from spleen, blood and lymph node in heterozygous mice. And there was no leaky expression of EGFP in other cell subpopulation.
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CD19+ cells depletion analysis
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Frequencies of CD19+; EGFP cells in spleen, blood and lymph node by flow cytometry. Splenocytes, blood and lymph node were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes, blood and lymph node was performed to assess the frequencies of CD19+; EGFP+ cells in total CD45+ cells. The signal for CD19+;EGFP+ cells in spleen(A), in blood (B) and lymph node(C) were significantly decreased in heterozygous mice injected with DT.
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CD19+ cells depletion analysis in spleen
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Frequencies of CD19+ cells and EGFP+ cells in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-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 CD19+ cells and EGFP+ cells in total CD45+ cells. The signal for CD19+ cells(A) and EGFP+ cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of CD19+ cells and EGFP+ cells in total CD45+ populations. The frequencies of CD19+ cells and EGFP+ cells were significantly decreased in heterozygous mice after DT injection.
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CD19+ cells depletion analysis in blood
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Frequencies of CD19+ cells and EGFP+ cells in blood by flow cytometry. Blood were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the blood was performed to assess the frequencies of CD19+ cells and EGFP+ cells in total CD45+ cells. The signal for CD19+ cells(A) and EGFP+ cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of CD19+ cells and EGFP+ cells in total CD45+ populations. The frequencies of CD19+ cells and EGFP+ cells were significantly decreased in heterozygous mice after DT injection.
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CD19+ cells depletion analysis in lymph node
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Frequencies of CD19+ cells and EGFP+ cells in lymph node by flow cytometry. Lymph node were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the lymph node was performed to assess the frequencies of CD19+ cells and EGFP+ cells in total CD45+ cells. The signal for CD19+ cells(A) and EGFP+ cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of CD19+ cells and EGFP+ cells in total CD45+ populations. The frequencies of CD19+ cells and EGFP+ cells were significantly decreased in heterozygous mice after DT injection.
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B cells depletion analysis
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Frequencies of B cells in spleen, blood and lymph node by flow cytometry. Splenocytes, blood and lymph node were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes, blood and lymph node was performed to assess the frequencies of B cells in total CD45+ cells. The signal for B cells in spleen(A), blood (B) and lymph node(C) were significantly decreased in heterozygous mice injected with DT.
Frequencies of EGFP in B cells from spleen, blood and lymph node by flow cytometry. Splenocytes, blood and lymph node were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old) injected with DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes, blood and lymph node was performed to assess the frequencies of EGFP cells in total B cells. The signal for EGFP in B cells in spleen(A), blood (B) and lymph node(C) were significantly decreased in heterozygous mice injected with DT.
Analysis of B cells depletion in spleen, blood and lymph node by flow cytometry. Splenocytes, blood and lymph node were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Cd19-EGFP-DTR-luciferase mice (Mut/+) (n=3, 6-7-week-old) injected with DT (50 ng per g body weight) for four consecutive days. The percentages of B cells in total CD45 cells from spleen, blood and lymph node(A) and EGFP+ cells in B cells from spleen, blood and lymph node(B) were significantly decreased in heterozygous mice injected with DT.
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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 B-Cd19-EGFP-DTR-luc mice(n=3, 6-7-week-old). Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in heterozygous B-Cd19-EGFP-DTR-luc mice were similar to those in C57BL/6 mice. The frequency of leukocyte subpopulations in blood and lymph node of B-Cd19-EGFP-DTR-luc mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM.
