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 L. Schnaar, Axel Roers, Karin Hartmann, Chang Min Lee, Delia Demers, Tao Zheng, Bruce Scott 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

Fingerprint

Sialic Acid Binding Immunoglobulin-like Lectins
Mast Cells
mice
acids
Acids
eosinophils
cytometry
Flow cytometry
Lectins
Eosinophils
allergic diseases
Flow Cytometry
Genes
Tissue
Cell Degranulation
leukocytes
kits
Basophils
bone marrow
stem cells

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, Yadong ; Chhiba, Krishan D. ; Zhang, Fengrui ; Ye, Xujun ; Wang, Lihui ; Zhang, Li ; Robida, Piper A. ; Moreno-Vinasco, Liliana ; Schnaar, Ronald L. ; Roers, Axel ; Hartmann, Karin ; Lee, Chang Min ; Demers, Delia ; Zheng, Tao ; Bochner, Bruce Scott ; Zhu, Zhou. / Mast cell-specific expression of human siglec-8 in conditional knock-in mice. In: International journal of molecular sciences. 2019 ; Vol. 20, No. 1.
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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.",
keywords = "Allergic disease, Mouse mast cells, ROSA26, Siglec-8",
author = "Yadong Wei and Chhiba, {Krishan D.} and Fengrui Zhang and Xujun Ye and Lihui Wang and Li Zhang and Robida, {Piper A.} and Liliana Moreno-Vinasco and Schnaar, {Ronald L.} and Axel Roers and Karin Hartmann and Lee, {Chang Min} and Delia Demers and Tao Zheng and Bochner, {Bruce Scott} and Zhou Zhu",
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Wei, Y, Chhiba, KD, Zhang, F, Ye, X, Wang, L, Zhang, L, Robida, PA, Moreno-Vinasco, L, Schnaar, RL, Roers, A, Hartmann, K, Lee, CM, Demers, D, Zheng, T, Bochner, BS & Zhu, Z 2019, 'Mast cell-specific expression of human siglec-8 in conditional knock-in mice' International journal of molecular sciences, vol. 20, no. 1, 19. https://doi.org/10.3390/ijms20010019

Mast cell-specific expression of human siglec-8 in conditional knock-in mice. / Wei, Yadong; Chhiba, Krishan D.; Zhang, Fengrui; Ye, Xujun; Wang, Lihui; Zhang, Li; Robida, Piper A.; Moreno-Vinasco, Liliana; Schnaar, Ronald L.; Roers, Axel; Hartmann, Karin; Lee, Chang Min; Demers, Delia; Zheng, Tao; Bochner, Bruce Scott; Zhu, Zhou.

In: International journal of molecular sciences, Vol. 20, No. 1, 19, 01.01.2019.

Research output: Contribution to journalArticle

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AU - Wei, Yadong

AU - Chhiba, Krishan D.

AU - Zhang, Fengrui

AU - Ye, Xujun

AU - Wang, Lihui

AU - Zhang, Li

AU - Robida, Piper A.

AU - Moreno-Vinasco, Liliana

AU - Schnaar, Ronald L.

AU - Roers, Axel

AU - Hartmann, Karin

AU - Lee, Chang Min

AU - Demers, Delia

AU - Zheng, Tao

AU - Bochner, Bruce Scott

AU - Zhu, Zhou

PY - 2019/1/1

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N2 - 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.

AB - 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.

KW - Allergic disease

KW - Mouse mast cells

KW - ROSA26

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