Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals.However,conventional strategy for producing tissue-specific knockout mice is a time- and labor-consumi...Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals.However,conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process,restricting rapid study of the gene function in vivo.CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique,which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes.Here,we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step.We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells.We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene.Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally.Consistently,male progeny from female founders were infertile and females could transmit the transgenes to the next generation.Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.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
Stem cells sustain the capacity of self-renewal and differentiation into specialized cells,offering exciting promises in both basic and applied research.As an example of an applied purpose,stem cells can be used as a ...Stem cells sustain the capacity of self-renewal and differentiation into specialized cells,offering exciting promises in both basic and applied research.As an example of an applied purpose,stem cells can be used as a resource in regenerative medicine for generation of appropriate展开更多
基金supported by the grants from the Ministry of Science and Technology of China(Nos.2014CB964803 and 2015AA020307)the National Natural Science Foundation of China(Nos.91319310,31225017 and 31530048)the Chinese Academy of Sciences(No.XDA01010403)
文摘Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals.However,conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process,restricting rapid study of the gene function in vivo.CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique,which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes.Here,we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step.We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells.We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene.Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally.Consistently,male progeny from female founders were infertile and females could transmit the transgenes to the next generation.Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.
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.
文摘Stem cells sustain the capacity of self-renewal and differentiation into specialized cells,offering exciting promises in both basic and applied research.As an example of an applied purpose,stem cells can be used as a resource in regenerative medicine for generation of appropriate
