An inducible mouse model of OI type V reveals aberrant osteogenesis caused by Ifitm5 c.-14C>T mutation

Osteogenesis imperfecta (OI) Type V is typically characterized by radial head dislocation, calcification of interosseous membrane and post-fracture hyperplastic callus. It is caused by the c.-14C>T mutation in the 5’UTR of the IFITM5 gene, adding five amino acids (MALEP) to the N-terminal of IFITM5 protein. Previous studies have suggested a neomorphic function of the MALEP-IFITM5 protein. However, the underlying mechanisms remain unclear due to embryonic lethality in previous models. Therefore, we developed an inducible animal model of Ifitm5flox c.-14C>T that could be induced by Cre expressing at different developmental stages to explore the pathogenic effects of the neomorphic MALEP-IFITM5. Specifically, Prx1-Cre; Ifitm5flox c.-14C>T mutant mice were born with fractures in all limbs, showing deficient ossification and enhanced chondrogenesis associated with increased SOX9 abundance. We isolated skeletal cells from tibia of Ifitm5 mutant mouse at P6 stage and conducted single RNA sequencing (10X Genomics). The data were compared to the RNA-seq data generated from the tibia of control mouse at the same stage (GSE159544). The protocol to isolate the mutant skeletal cells for single cell RNA sequencing was the same as the control sample (GSE159544). Right and left tibia from Prx1-Cre; Ifitm5flox c.-14C>T mutant mouse at P6 stage were dissected with soft tissues removed. Bone tissue was cut into small pieces and blood cells were removed with TrypLE Express digession for 10 minutes at 37 degree. The remaining bone tissue was digested with type II collagenase (0.25%) and Dispase (0.25%) in HBSS for 1.5 hours. Cells were collected every half an hour, and digestion medium was changed each time. Red blood cells were lysised and dead cells were removed using kits. The remaining cells were tested for cell viability (>80%). Single cells were encapsulated for library preparation and sequencing using the Chromium single cell platform (10X Genomics Inc)according to the manufacturer’s protocol. Library size and concentration were determined by Qubit, quantitative PCR, and Bioanalyzer assays. A raw total input of 15000 cells was estimated for each sample. Library preparation and Illumina (NovaSeq 6000) sequencing (151 bp paired end) were performed at HKU CPOS, at 150 Gbp throughput per sample.