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Int. J. Mol. Sci. 2019, 20(1), 19; https://doi.org/10.3390/ijms20010019

Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

1
Section of Allergy and Clinical Immunology, Yale University School of Medicine, New Haven, CT 06511, USA
2
Department of Medicine, Division of Allergy and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
3
Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
4
Institute of Immunology, University of Technology Dresden, 01069 Dresden, Germany
5
Department of Dermatology, University of Lübeck, 23538 Lübeck, Germany
6
Department of Molecular Microbiology and Immunology, Department of Pediatrics, Brown University Alpert Medical School, Providence, RI 02912, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Received: 26 November 2018 / Revised: 4 December 2018 / Accepted: 14 December 2018 / Published: 21 December 2018
(This article belongs to the Special Issue Glycan–Receptor Interaction 2018)
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Abstract

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo. View Full-Text
Keywords: allergic disease; mouse mast cells; ROSA26; Siglec-8 allergic disease; mouse mast cells; ROSA26; Siglec-8
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Wei, Y.; Chhiba, K.D.; Zhang, F.; Ye, X.; Wang, L.; Zhang, L.; Robida, P.A.; Moreno-Vinasco, L.; Schnaar, R.L.; Roers, A.; Hartmann, K.; Lee, C.-M.; Demers, D.; Zheng, T.; Bochner, B.S.; Zhu, Z. Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice. Int. J. Mol. Sci. 2019, 20, 19.

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