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. 2018 Jun 29;1:83. doi: 10.1038/s42003-018-0081-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Eosinophil-specific VAMP-7 gene deletion in eoCRE/V7 mice confirmed by qPCR. a VAMP-7 gene deletion protocol used to generate eoCRE/V7 offspring. Amplicon values obtained from eoCRE/V7 offspring were compared to control VAMP-7^f/f mice to generate ΔCt and ΔΔCt ratios as described in Methods section. Diagram adapted from Sato et al.³². b qPCR-quantified VAMP-7 gene deletion in bone marrow-derived eosinophils isolated from Zp3/V7, VAMP-7^f/f, eoCRE^+/−, and eoCRE/V7 mice. Low ΔΔCt values indicate VAMP-7 gene deletion. All data were normalized to VAMP-7^f/f values. Data are presented as mean ± min-max range. c VAMP-7 gene excision efficiency between eosinophils and lymphocytes isolated from eoCRE/V7/I5 mice. Peripheral blood eosinophils (CCR3⁺/IL-5Rα⁺/Gr-1⁺/CD4⁻/B220⁻) and lymphocytes (CCR3⁻/IL-5Rα⁻/Gr-1⁻/CD4⁺/B220⁺) were analyzed from Zp3/V7 or eoCRE/V7/I5 mice. All data were normalized to the average ΔΔCt value for the eoCRE/V7/I5 lymphocyte population. Closed symbols indicate eoCRE^+/− mice, open symbols indicate eoCRE/V7 mice. d VAMP-7-specific gene deletion was restricted to eoCRE/V7/I5-derived eosinophils. The P-a and P-b primer pair shown in (a) was used to detect expression of the deleted gene sequence (326 bps) in both eosinophils (CCR3⁺/IL-5Rα⁺/Gr-1⁺/CD4⁻/B220⁻) and lymphocytes (CCR3⁻/IL-5Rα⁻/Gr-1⁻/CD4⁺/B220⁺) isolated from eoCRE/V7/I5 mice. Expression profiles of the latter strain were compared to Zp3/V7/I5 whole-blood leukocytes, eoCRE/I5 eosinophils, and I5 eosinophils. e Western blot analysis of VAMP-7 protein expression in eoCRE/V7/I5-derived eosinophils (~96% purity), as well as in bone marrow-and peripheral blood-derived eosinophils from eoCRE/I5 mice. Gel was cropped to improve conciseness of presentation. f peripheral blood WBC profiles of eoCRE^+/− and eoCRE/V7 mice. g Transmission electron microscopy images of peripheral blood eosinophils from eoCRE/I5 and eoCRE/V7/I5 mice. Scale bar, 1 μm. h EPX levels in BAL fluid from I5/hE2/eoCRE^+/− and I5/hE2/eoCRE/V7 mice. For Fig. 2b, c, f, and h, n = 3–7 mice/experiment. Data are presented as mean ± SEM. For Fig. 2d, e, and g, one representative gel, blot, or image from three separate experiments is shown. **p < 0.01 (one-way ANOVA, Tukey’s)