GSE117643
Gene expression profiles of differentiating BMSCs from WT and Irx5 knockout mice during osteoblastogenesis (day 4 post differentiation)
Functional mutagenic screening has been used to identify key factors required for a wide range of developmental processes. However, such screens can only be successfully applied in lower organisms. Here, through combined application of androgenetic haploid embryonic stem cells (haESCs) and CRISPR-Cas9-based genome editing, we quickly identify pivotal factors involved in early bone development from a medium-scale gene library. Injection of haESCs carrying constitutively expressed Cas9 and an sgRNA library containing 216 sgRNAs targeting 72 preselected candidate genes into oocytes produced biallelic mutant mice with high efficiency. Through skeletal phenotype analysis by Alcian blue and Alizarin Red staining at birth, we identified transcriptional factor Irx5 as a positive regulator of bone development. Irx5 KO mice generated by regular method exhibited obvious decreased bone mass and increased adipogenesis in the bone marrow. Moreover, the deletion of Irx5 from bone marrow cells prohibited osteoblast differentiation and enhanced adipocyte differentiation ability in vitro. Mechanistically, IRX5 promotes osteoblastogenesis and inhibits adipogenesis by suppressing PPAR? expression. We conclude that haESC-mediated SC technology, in combination with a targeted sgRNA library, can be used as a powerful reverse genetic screening strategy to identify pivotal factors involved in mouse development. Overall design: Gene expression examination of BMSCs from WT and Irx5 knockout mice after 4 days osteoblast differentiation.Each sample has 3 repeats.