Construction of Fzd6Q152Emice through CRISPR/Cas9 technology and their reproduction and identification.

Abstract

The CRISPR/Cas9 system is widely used for genome editing in human, rat and mouse cells. In this study, we established Fzd6 mutant mice using CRISPR/Cas9 technology, and obtained Fzd6 homozygous mutant (Fzd6Q152E) mice through breeding. Fzd6 plays a role in depression, but there are few related reports. We used this model to investigate the mechanism of Fzd6 involved in depression, and build a solid foundation for subsequent in-depth studies. The target of Fzd6 mutation was obtained by CRISPR/Cas9 technology and hippocampal tissue was collected for Nissl staining and histological analysis. Blood was collected for enzyme linked immunosorbent assay (ELISA); The gene expression of Fzd6 and the related genes expression in wnt pathway was quantified by quantitative real-time PCR (qRT-PCR), and then expression of Fzd6 and proteins in the Wnt pathway were identified by western blotting. ELISA results showed that the expression levels of brain derived neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT), and Noradrenaline (NE) in serum were significantly decreased in Fzd6Q152E mice, whereas the mRNA expression of Lrp5, Lrp6, and Dkk2 is increased. The western blotting revealed that the expression of Fzd6 and Lrp6 is decreased, although the expression of Dkk2 and Gsk-3β increased. Our study successfully established homozygous Fzd6 mutant mice model. The relationship between Fzd6-Wnt and depression was preliminarily clarified, which provides an ideal animal model for subsequent research on diseases induced by the Fzd6 mutation.