Figure 1. Generation and characterization of Prtn3^–/–Ela2^–/– mice.

(A) Southern blot showing the WT (+/+) 11.5-kb band of the nontargeted allele and the presence of an additional 7.5-kb band in a heterozygous (+/–) PR3/NE-targeted embryonic stem cell clone. (B) RT-PCR revealed the complete lack of mouse PR3 (mPR3; 437 bp) and mouse NE (743 bp) transcripts in bone marrow cells from Prtn3^–/–Ela2^–/– mice (–/–), while expression of β-actin (699 bp) was normal. (C) Casein zymography showed prominent casein degradative activity at 27 kDa in WT neutrophil lysates, while intermediate degradation by heterozygous lines and no degradation by Prtn3^–/–Ela2^–/– lines was found at this size. M, marker. (D) Western blot analysis of granule enzyme expression in bone marrow–derived neutrophils. In Prtn3^–/–Ela2^–/– neutrophils, no signals for PR3 and NE were detected, while CG (~26 kDa), MPO (~59 kDa), and a smaller degradation product of MPO were detected at the same levels as in WT neutrophils. (E) Microscopic analysis of H&E-stained blood smears revealed normal granulocyte morphology in Prtn3^–/–Ela2^–/– mice, with a polymorphic nucleus (dark blue) identical to that of WT neutrophils. Original magnification, ×20. (F) Flow cytometry of peripheral blood with gating on Gr-1^hiCD11b⁺ showed regular neutrophil populations (boxed regions) in Prtn3^–/–Ela2^–/– mice. Plots are representative of data obtained from 3 mice per group. Percentages denote percent cells in the boxed regions.