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.
Highlights
Sialic acid immunoglobulin-like lectin 8 (Siglec-8) is a member of the CD33 subfamily of glycan receptors called Siglecs
To verify that the Neo-STOP fragment in these constructs can be removed by Cre, and that the Siglec-8 cDNA gets appropriately expressed on the cell surface, we separately transfected the two constructs into confluent HEK 293T cells with or without co-transfection of the pCAG-Cre:Green fluorescence protein (GFP) plasmid using Lipofectamine 2000
These results demonstrated that: (1) The ROSA26 vector-based conditional Siglec-8 targeting constructs were responsive to Cre; (2) Siglec-8 can be conditionally expressed on the cell surface; and (3) between the two constructs, the pCAG-Neo-Siglec-8 construct was more efficient
Summary
Sialic acid immunoglobulin-like lectin 8 (Siglec-8) is a member of the CD33 subfamily of glycan receptors called Siglecs. Siglec-8 is unique in its expression on human eosinophils, mast cells, and basophils, which are important innate immune effector cells in allergy, asthma, and other type 2 disorders [1,2,3,4]. Siglec-F on eosinophils is considered a functional paralog of Siglec-8 because of some similarities in expression patterns, recognition of glycan ligands, and regulatory functions [11,12]. There are some noticeable differences between Siglec-8 and Siglec-F, including patterns of expression on other cells (e.g., Siglec-F is not expressed by mouse mast cells), additional sialylated, non-sulfated ligands recognized by Siglec-F, and more modest death responses seen upon Siglec-F engagement [3,13]
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