Basic Information
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Targeting strategy
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Gene targeting strategy for B-Ncr1-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 Ncr1 gene in B-Ncr1-EGFP-DTR-luciferase mice, allowing for EGFP, DTR and luciferase expression and de novo Ncr1 expression. The coding sequences were separated by self-cleaving 2A peptide sequences.
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NK cells depletion analysis in spleen
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Frequencies of NK cells and EGFP+ cells in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Ncr1 EGFP-DTR-luciferase mice (Mut/+) (n=3, 8-9-week-old) injected with PBS or DT (50 ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequencies of EGFP+ cells and NK cells in total CD45+ cells. The signal for NK cells(A) and EGFP+ cells(B) were decreased in heterozygous mice injected with DT. C. Percentages of NK cells and EGFP+ cells in total CD45+ populations. The frequencies of NK cells and EGFP+ cells were decreased in heterozygous mice after DT injection.
Frequency of EGFP+ cells in NK cells from spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-Ncr1-EGFP-DTR-luciferase mice (Mut/+) (n=3, 8-9-week-old) injected with PBS or 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 NK cells(A). The percentage of EGFP+ in NK cells(B) was 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 C57BL/6 and B-Ncr1-EGFP-DTR-luc mice (n=3, 9-week-old). Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. Percentages of T cell, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in heterozygous B-Ncr1-EGFP-DTR-luc mice were similar to those in the C57BL/6 mice. The frequency of leukocyte subpopulations in blood and lymph node of B-Ncr1-EGFP-DTR-luc mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM.
