Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development.
Meizhu BaiYujiao HanYuxuan WuJiaoyang LiaoLin LiLijun WangQing LiWenhui XingLuonan ChenWeiguo ZouJinsong LiPublished in: PLoS biology (2019)
Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- mice exhibited small skeletal size only at birth, Irx5-/- mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.
Keyphrases
- genome wide
- high fat diet induced
- embryonic stem cells
- crispr cas
- genome editing
- bone marrow
- bone mineral density
- insulin resistance
- wild type
- cancer therapy
- gene expression
- quantum dots
- type diabetes
- metabolic syndrome
- preterm infants
- bone loss
- drug delivery
- postmenopausal women
- binding protein
- genome wide identification
- highly efficient
- preterm birth
- platelet rich plasma
- bioinformatics analysis
- genome wide analysis
- fatty acid