B-Ncr1-EGFP-DTR-luciferase mice have the endogenous mouse Ncr1 promoter to determine the expression of DTR (Diphtheria toxin receptor), EGFP (enhanced green fluorescent protein) and luciferase.
NK cells were traced by EGFP in B-Ncr1-EGFP-DTR-luciferase mice, and could be effectively eliminated after DT administration.
Targeting strategy
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.
Frequency of leukocyte subpopulations in spleen
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.
NK cells depletion analysis in spleen
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(v2) (Mut/+) (n=3, 8-9-week-old) injected with PBS or DT (50ng per g body weight) for four consecutive days. Flow cytometry analysis of the splenocytes was performed to assess the frequency of EGFP+ cells in NK cells. The frequency of EGFP in NK cells was decreased in heterozygous mice after DT injection.