• 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.

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 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. 

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.