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通過單倍體胚胎干細胞在小鼠中進行靶向基因篩選，確定骨發育中的關鍵基因

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發表時間：2024-09-04 15:58

2019年7月，細胞生物學國家重點實驗室；中國科學院上海生物化學與細胞生物學研究所；中國科學院分子細胞科學**中心；上海分子男科重點實驗室；中國科學院上海生物化學與細胞生物學研究所；中國科學院分子細胞科學**中心；細胞生物學國家重點實驗室；中國科學院大學；華東師范大學生物醫學科學研究所和生命科學學院上海市調控生物學重點實驗室；中國科學院上海生物科學研究院生物化學與細胞生物學研究所；中科院分子細胞科學**中心；細胞信號網絡創新中心；系統生物學重點實驗室；生命科學與技術學院；上海工業大學 (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老師研究團隊在《PLOS Biology》上發表論文：

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

“通過單倍體胚胎干細胞在小鼠中進行靶向基因篩選，確定骨發育中的關鍵基因”

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：

誘變篩選對于識別參與發育過程的關鍵基因是強有力的。然而，這種篩選只在低等生物中成功。在這里，科研人員通過結合雄激素單倍體胚胎干細胞(AG-haESCs)和聚集規律間隔回復性重復序列/ crispr相關蛋白9 (CRISPR-Cas9)技術，在小鼠中開發了一種靶向遺傳篩選方法。科研人員通過卵母細胞注射AG-haESCs產生突變型半克隆(SC)小鼠池，其中AG-haESCs攜帶組成型表達Cas9和單個引導RNA (sgRNA)文庫，一次性靶向72個預先選擇的基因，并通過出生時骨骼分析篩選骨骼發育相關基因。這產生了4個基因:Zic1和Clec11a，這是骨骼發育所必需的，以及Rln1和Irx5，這是以前沒有考慮過的。Rln1?/?小鼠僅在出生時表現出較小的骨骼尺寸，而Irx5?/?小鼠由于骨量減少和骨髓脂肪生成增加而在出生后和成人期均表現出骨骼異常。iroquois homeobox 5 (IRX5)通過抑制過氧化物酶體增殖物激活受體γ (PPARγ)的激活，促進成骨細胞的形成，抑制脂肪的形成。因此，ag - haesc介導的功能性誘變篩選為小鼠發育過程的遺傳詢問開辟了新的途徑。