Generation and phenotype analysis of CysLTR1 L118F mutant mice.

Abstract

Cysteinyl leukotrienes (CysLTs) are a group of eicosanoids that regulate the pathogenesis of various human diseases, mainly by signaling through the cysteinyl leukotriene receptor 1 (CysLTR1). The aim of this study was to generate and examine the phenotype of CysLTR1 L118F mutant mice. CysLTR1 L118F mutant mice were generated by the simultaneous microinjection of single guide RNA, Cas9 messenger RNA, and donor plasmid into fertilized mouse eggs. The morphological and behavioral characteristics of the resultant CysLTR1 L118F mutant mice were analyzed using an animal phenotype analysis platform, which included the assessment of body length, tail length, grip strength, and locomotor activity. Immunoprecipitation coupled with mass spectrometry was performed to identify CysLTR1-interacting proteins, and the intracellular calcium levels were determined using fluorometric imaging plate reader assays. The body length and tail length of CysLTR1 L118F mutant mice were significantly increased compared with wild-type mice. In addition, the grip strength and locomotor activity were remarkably elevated in L118F mutant mice compared with wild-type mice. Only three proteins were found to interact with both wild-type and CysLTR1 L118F proteins, whereas 4 and 13 additional proteins interacted exclusively with wild-type and mutant CysLTR1, respectively. Lastly, the responsiveness of cardiac muscle cells to CysLTs were significantly impaired by the L118F substitution in CysLTR1 proteins. The CysLTR1 L118F point mutation induced significant changes in the mouse morphology and behavior, which might be mediated by alterations of its protein interaction profile.