Mast cell-specific expression of human siglec-8 in conditional knock-in mice

Yadong Wei, Krishan D. Chhiba, Fengrui Zhang, Xujun Ye, Lihui Wang, Li Zhang, Piper A. Robida, Liliana Moreno-Vinasco, Ronald Lee Schnaar, Axel Roers, Karin Hartmann, Chang Min Lee, Delia Demers, Tao Zheng, Bruce S. Bochner, Zhou Zhu

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number19
JournalInternational Journal of Molecular Sciences
Volume20
Issue number1
DOIs
StatePublished - Jan 1 2019

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Keywords

  • Allergic disease
  • Mouse mast cells
  • ROSA26
  • Siglec-8

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

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. (2019). Mast cell-specific expression of human siglec-8 in conditional knock-in mice. International Journal of Molecular Sciences, 20(1), [19]. https://doi.org/10.3390/ijms20010019