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通过单倍体胚胎干细胞在小鼠中进行靶向基因筛选，确定骨发育中的关键基因

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发表时间：2024-09-04 15:58

2019年7月，细胞生物学国家重点实验室；中国科学院上海生物化学与细胞生物学研究所；中国科学院分子细胞科学**中心；上海分子男科重点实验室；中国科学院上海生物化学与细胞生物学研究所；中国科学院分子细胞科学**中心；细胞生物学国家重点实验室；中国科学院大学；华东师范大学生物医学科学研究所和生命科学学院上海市调控生物学重点实验室；中国科学院上海生物科学研究院生物化学与细胞生物学研究所；中科院分子细胞科学**中心；细胞信号网络创新中心；系统生物学重点实验室；生命科学与技术学院；上海工业大学 (State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China；State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China；Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China；Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science,Innovation Center for Cell Signaling Network, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai, China；School of Life Science and Technology, Shanghai Tech University, Shanghai, China) Jinsong Li老师研究团队在《PLOS Biology》上发表论文：

“Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development”

“通过单倍体胚胎干细胞在小鼠中进行靶向基因筛选，确定骨发育中的关键基因”

Abstract：

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.

摘要：

诱变筛选对于识别参与发育过程的关键基因是强有力的。然而，这种筛选只在低等生物中成功。在这里，科研人员通过结合雄激素单倍体胚胎干细胞(AG-haESCs)和聚集规律间隔回复性重复序列/ crispr相关蛋白9 (CRISPR-Cas9)技术，在小鼠中开发了一种靶向遗传筛选方法。科研人员通过卵母细胞注射AG-haESCs产生突变型半克隆(SC)小鼠池，其中AG-haESCs携带组成型表达Cas9和单个引导RNA (sgRNA)文库，一次性靶向72个预先选择的基因，并通过出生时骨骼分析筛选骨骼发育相关基因。这产生了4个基因:Zic1和Clec11a，这是骨骼发育所必需的，以及Rln1和Irx5，这是以前没有考虑过的。Rln1−/−小鼠仅在出生时表现出较小的骨骼尺寸，而Irx5−/−小鼠由于骨量减少和骨髓脂肪生成增加而在出生后和成人期均表现出骨骼异常。iroquois homeobox 5 (IRX5)通过抑制过氧化物酶体增殖物激活受体γ (PPARγ)的激活，促进成骨细胞的形成，抑制脂肪的形成。因此，ag - haesc介导的功能性诱变筛选为小鼠发育过程的遗传询问开辟了新的途径。