利用腺病毒递送系统，用靶向Orai的短发夹RNA（shRNAs）或表达显性负性Orai通道突变的cDNA转导细胞，对人肥大细胞内源性钙释放激活钙通道（CRAC通道）进行研究。

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.

作者信息

Ashmole Ian, Bradding Peter

机构信息

Department of Infection, Immunity and Inflammation, Institute for Lung Heath, University of Leicester, Glenfield Hospital, Leicester, UK.

Department of Respiratory Medicine, Institute for Lung Heath, University of Leicester, Glenfield Hospital, Leicester, UK.

出版信息

Methods Mol Biol. 2018;1843:115-124. doi: 10.1007/978-1-4939-8704-7_10.

DOI:10.1007/978-1-4939-8704-7_10

PMID:30203282

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.

摘要

我们描述了两种研究人肺肥大细胞中CRAC通道功能的方法。这两种方法都涉及对内源通道功能的抑制。第一种方法是我们使用靶向Orai的短发夹RNA（shRNAs）来敲低Orai通道mRNA转录本。第二种方法是我们过表达Orai通道家族三个成员的显性负性突变体。为了克服肥大细胞转染效率低的问题，我们采用腺病毒递送系统进行细胞转导。最初使用定量逆转录聚合酶链反应（RT-PCR）评估CRAC通道转录本的敲低情况。我们描述了一种用于检测β-己糖胺酶释放的试验，以此作为肥大细胞脱颗粒的指标，来评估显性负性突变体过表达的效果。