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Exon Skipping of FcεRIβ for Allergic Diseases

  • Greer K. Arthur
  • Glenn CruseEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1828)

Abstract

Mast cells are key effector cells in allergic inflammation and consequently are ideal targets for new therapeutics. The high-affinity IgE receptor complex, FcεRI, plays a critical role in mast cell and basophil activation by allergens to drive the immediate allergic inflammatory response. The β subunit of FcεRI is critical for trafficking the FcεRI complex to the cell membrane and amplifies the FcεRI signaling cascade. We have utilized splice switching antisense oligonucleotides to force expression of a truncated isoform of FcεRIβ, which we have shown does not associate with the FcεRI complex. This approach eliminates surface FcεRI expression in mast cells by targeting protein–protein interactions. Exon skipping has several therapeutic applications, and our findings demonstrate a novel application to alter receptor trafficking and dampen allergic inflammation. Here, we describe the methods of exon skipping in mast cells and the assays used to examine the responses of mast cells in vitro and in vivo.

Key words

Mast cell Allergic diseases Allergic inflammation FcεRI Exon skipping 

Abbreviations

AON

Antisense oligonucleotide

APC

Allophycocyanin

BMMC

Bone marrow-derived mast cells

BSA

Bovine serum albumin

dH2O

Deionized H2O

DMSO

Dimethylsulfoxide

DNP-HSA

Dinitrophenyl-human serum albumin

FcεRI

High-affinity IgE receptor

FcεRIβ

β subunit of the FcεRI complex

FITC

Fluorescein isothiocyanate

PBS

Phosphate-buffered saline

PE

Phycoerythrin

PNAG

p-nitrophenyl N-acetyl-β-D-glucosamide

rhSCF

Recombinant human stem cell factor

t-FcεRIβ

Truncated variant of FcεRIβ lacking exon 3

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Molecular Biomedical Sciences, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA

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