Establishment of a CRISPR/Cas9-Mediated Cysltr1 Knockout Mouse Model and iTRAQ-Based Proteomic Analysis

Abstract

Purpose: To clarify the role of Cysteinyl leukotrienes receptor type 1 (CYSLTR1) and find the potential predictors of CYSLTR1 antagonists (leukotriene receptor antagonists [LTRAs]) responsiveness in vivo.

Experimental design: Cysltr1 knockout (KO) mouse model is established by the CRISPR/Cas9 system. The phenotype of Cysltr1 KO mice are tested by western blotting (WB), histological examinations, and experiment of zymosan A-induced peritoneal inflammation. The differentially expressed proteins (DEPs) between the Cysltr1 KO and the wild type (WT) mice lung tissues are analyzed by the iTRAQ-based proteomic technology. WB is used to validate a subset of DEPs. The total nitric oxide (NO) concentration in lung tissues are measured.

Results: The Cysltr1 KO mice show the decrease of vascular permeability in comparison with the WT mice. Our quantitative proteomic analysis identified 239 DEPs in total. WB confirms an increased expression of protein kinase C-δ (PKC-δ), while N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1) and β-Catenin expression are reduced. The total NO concentrations are significantly reduced in lungs from Cysltr1 KO mice.

Conclusions and clinical relevance: This study not only provides a comprehensive dataset on overall protein changes in Cysltr1 KO mice lung tissues, but also sheds light on interpreting the description of lower vascular permeability in Cysltr1 KO mice.

Keywords: CRISPR/Cas9; cysteinyl leukotrienes receptor; iTRAQ.