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Study of Endogenous CRAC Channels in Human Mast Cells Using an Adenoviral Delivery System to Transduce Cells with Orai-Targeting shRNAs or with cDNAs Expressing Dominant-Negative Orai Channel Mutations

  • Ian Ashmole
  • Peter Bradding
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1843)

Abstract

We describe two methods to study CRAC channel function in human lung mast cells. Both methods involve suppression of endogenous channel function. In the first we use Orai-targeting shRNAs to knock down Orai channel mRNA transcripts. In the second we overexpress dominant-negative mutants of the three members of the Orai channel family. To overcome the poor transfection efficiency of mast cells, we employ an adenoviral delivery system for cell transduction. Knockdown of CRAC channel transcripts is assessed initially using quantitative RT-PCR. We describe an assay for β-hexosaminidase release as a measure of mast cell degranulation to assess the effect of overexpression of dominant-negative mutants.

Key words

Mast cells RNA interference (RNAiShort hairpin RNA (shRNA) Adenovirus Transduction Dominant-negative mutant Degranulation 

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Copyright information

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

Authors and Affiliations

  • Ian Ashmole
    • 1
    • 2
  • Peter Bradding
    • 1
    • 2
  1. 1.Department of Infection, Immunity and Inflammation, Institute for Lung HeathUniversity of Leicester, Glenfield HospitalLeicesterUK
  2. 2.Department of Respiratory Medicine, Institute for Lung HeathUniversity of Leicester, Glenfield HospitalLeicesterUK

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