With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Rece...With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell （haESC） approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches.展开更多
Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate th...Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches.Recently,mouse haploid embryonic stem(haES)cells have been successfully isolated from parthenogenetic and androgenetic embryos,providing an ideal tool for genetic analyses.In these studies,mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination.In particular,haES cells from androgenetic embryos can be employed as novel,renewable form of fertilization agent for yielding live-born mice via injection into oocytes,thus showing the possibility that genetic analysis can be extended from cellular level to organism level.展开更多
Heparan sulfate proteoglycan 2(HSPG2)gene encodes the matrix protein Perlecan,and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects(NTDs).We discovered rare genet...Heparan sulfate proteoglycan 2(HSPG2)gene encodes the matrix protein Perlecan,and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects(NTDs).We discovered rare genetic variants of HSPG2 in 10%cases compared to only 4%in controls among a cohort of 369 NTDs.Endorepellin,a peptide cleaved from the domain V of Perlecan,is known to promote angiogenesis and autophagy in endothelial cells.The roles of enderepellin in neurodevelopment remain unclear so far.Our study revealed that endorepellin can migrate to the neuroepithelial cells and then be recognized and bind with the neuroepithelia receptor neurexin in vivo.Through the endocytic pathway,the interaction of endorepellin and neurexin physiologically triggers autophagy and appropriately modulates the differentiation of neural stem cells into neurons as a blocker,which is necessary for normal neural tube closure.We created knock-in(KI)mouse models with human-derived HSPG2 variants,using sperm-like stem cells that had been genetically edited by CRISPR/Cas9.We realized that any HSPG2 variants that affected the function of endorepellin were considered pathogenic causal variants for human NTDs given that the severe NTD phenotypes exhibited by these KI embryos occurred in a significantly higher response frequency compared to wildtype embryos.Our study provides a paradigm for effectively confirming pathogenic mutations in other genetic diseases.Furthermore,we demonstrated that using autophagy inhibitors at a cellular level can repress neuronal differentiation.Therefore,autophagy agonists may prevent NTDs resulting from failed autophagy maintenance and neuronal over-differentiation caused by deleterious endorepellin variants.展开更多
小鼠孤雄单倍体胚胎干细胞携带一套来源于精子的遗传物质,与二倍体胚胎干细胞相似,拥有干细胞的自我更新和多向分化等特性,能够在体外长期稳定地培养。重要的是,该细胞能够代替精子在注入到卵母细胞中后,产生半克隆小鼠。因其兼具干细...小鼠孤雄单倍体胚胎干细胞携带一套来源于精子的遗传物质,与二倍体胚胎干细胞相似,拥有干细胞的自我更新和多向分化等特性,能够在体外长期稳定地培养。重要的是,该细胞能够代替精子在注入到卵母细胞中后,产生半克隆小鼠。因其兼具干细胞和精子特性,故又被称为“人造精子”细胞或者类精子干细胞。将孤雄单倍体胚胎干细胞与CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)等新型基因编辑技术结合起来,能够在细胞水平和动物个体水平进行精准、复杂和高通量的遗传改造,为基因的功能研究提供了强有力的工具。本文综述了孤雄单倍体胚胎干细胞的建立,及其在印记基因功能研究、遗传筛选、人类疾病模拟和蛋白质标签打靶等方面的应用。展开更多
基金supported by the National Natural Science Foundation of China(3731530048C1202)
文摘With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell （haESC） approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches.
基金supported by grants from the Ministry of Science and Technology of China(No.2009CB941101 to J.L.)the "Strategic Priority Research Program" of the Chinese Academy of Sciences(No.XDA01010403 to J.L.).
文摘Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches.Recently,mouse haploid embryonic stem(haES)cells have been successfully isolated from parthenogenetic and androgenetic embryos,providing an ideal tool for genetic analyses.In these studies,mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination.In particular,haES cells from androgenetic embryos can be employed as novel,renewable form of fertilization agent for yielding live-born mice via injection into oocytes,thus showing the possibility that genetic analysis can be extended from cellular level to organism level.
基金supported by the National Key Research and Development Program of China(2021YFC2701100)the National Natural Science Foundation of China(81930036,32293230 and 8215008)+1 种基金the Commission for Science and Technology of Shanghai Municipality(20JC1418500 and 20ZR1404800)Project supported by Shanghai Municipal Science and Technology Major Project。
文摘Heparan sulfate proteoglycan 2(HSPG2)gene encodes the matrix protein Perlecan,and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects(NTDs).We discovered rare genetic variants of HSPG2 in 10%cases compared to only 4%in controls among a cohort of 369 NTDs.Endorepellin,a peptide cleaved from the domain V of Perlecan,is known to promote angiogenesis and autophagy in endothelial cells.The roles of enderepellin in neurodevelopment remain unclear so far.Our study revealed that endorepellin can migrate to the neuroepithelial cells and then be recognized and bind with the neuroepithelia receptor neurexin in vivo.Through the endocytic pathway,the interaction of endorepellin and neurexin physiologically triggers autophagy and appropriately modulates the differentiation of neural stem cells into neurons as a blocker,which is necessary for normal neural tube closure.We created knock-in(KI)mouse models with human-derived HSPG2 variants,using sperm-like stem cells that had been genetically edited by CRISPR/Cas9.We realized that any HSPG2 variants that affected the function of endorepellin were considered pathogenic causal variants for human NTDs given that the severe NTD phenotypes exhibited by these KI embryos occurred in a significantly higher response frequency compared to wildtype embryos.Our study provides a paradigm for effectively confirming pathogenic mutations in other genetic diseases.Furthermore,we demonstrated that using autophagy inhibitors at a cellular level can repress neuronal differentiation.Therefore,autophagy agonists may prevent NTDs resulting from failed autophagy maintenance and neuronal over-differentiation caused by deleterious endorepellin variants.
文摘小鼠孤雄单倍体胚胎干细胞携带一套来源于精子的遗传物质,与二倍体胚胎干细胞相似,拥有干细胞的自我更新和多向分化等特性,能够在体外长期稳定地培养。重要的是,该细胞能够代替精子在注入到卵母细胞中后,产生半克隆小鼠。因其兼具干细胞和精子特性,故又被称为“人造精子”细胞或者类精子干细胞。将孤雄单倍体胚胎干细胞与CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)等新型基因编辑技术结合起来,能够在细胞水平和动物个体水平进行精准、复杂和高通量的遗传改造,为基因的功能研究提供了强有力的工具。本文综述了孤雄单倍体胚胎干细胞的建立,及其在印记基因功能研究、遗传筛选、人类疾病模拟和蛋白质标签打靶等方面的应用。
