CRISPR/Cas9[Clustered regular interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9]系统是广泛存在于细菌和古生菌中可降解入侵病毒和噬菌体DNA的一种基因定点编辑技术,由于其具有快速、精准、高效,易于设计...CRISPR/Cas9[Clustered regular interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9]系统是广泛存在于细菌和古生菌中可降解入侵病毒和噬菌体DNA的一种基因定点编辑技术,由于其具有快速、精准、高效,易于设计,且特异性高等优势,得到了广泛的应用。概述了CRISPR/Cas9基因编辑技术的发展历程,并介绍其结构和编辑原理、在动物细胞系构建中的应用及展望,以便为更合理地应用和改进该技术提供系统的文献参考。展开更多
We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA f...We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.展开更多
Members of the basic helix-loop-helix （bHLH） gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer （FRET） is used to monitor b...Members of the basic helix-loop-helix （bHLH） gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer （FRET） is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD 1, Mash 1, Neurogenin 1 （Ngn 1）, Neurogenin2 （Ngn2）, and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein （EYFP） and enhanced cyan fluorescence protein （ECFP）, respectively. The subcellular localization and mobility ofbHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mashl/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.展开更多
As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expre...As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.展开更多
Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell prot...Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.展开更多
中国科学院生态环境研究中心汪海林研究团队在生物分析与表观遗传领域取得新进展。该研究在哺乳动物细胞系中检测到的DNA N^6-甲基腺嘌呤(6mA)并不依赖于甲基化转移酶,并发现了DNA聚合酶依赖的DNA 6mA新来源。相关研究工作以“N^6-methy...中国科学院生态环境研究中心汪海林研究团队在生物分析与表观遗传领域取得新进展。该研究在哺乳动物细胞系中检测到的DNA N^6-甲基腺嘌呤(6mA)并不依赖于甲基化转移酶,并发现了DNA聚合酶依赖的DNA 6mA新来源。相关研究工作以“N^6-methyladenine is incorporated into mammalian genome by DNA polymerase”(N^6-甲基腺嘌呤由DNA聚合酶掺入哺乳动物基因组)为题于2020年4月30日在线发表于Cell Research(https://doi.org/10.1038/s41422-020-0317-6)。展开更多
All eukaryotic genomes have genes with introns in variable sizes.As far as spliceosomal introns are concerned,there are at least three basic parameters to stratify introns across diverse eukaryotic taxa:size,number,an...All eukaryotic genomes have genes with introns in variable sizes.As far as spliceosomal introns are concerned,there are at least three basic parameters to stratify introns across diverse eukaryotic taxa:size,number,and sequence context.The number parameter is highly variable in lower eukaryotes,especially among protozoan and fungal species,which ranges from less than4%to 78%of the genes.Over greater evolutionary time scales,the number parameter undoubtedly increases as observed in higher plants and higher vertebrates,reaching greater than 12.5 exons per gene in average among mammalian genomes.The size parameter is more complex,where multiple modes appear at work.Aside from intronless genes,there are three other types of intron-containing genes:half-sized,minimal,and size-expandable introns.The half-sized introns have only been found in a limited number of genomes among protozoan and fungal lineages and the other two types are prevalent in all animal and plant genomes.Among the size-expandable introns,the sizes of plant introns are expansion-limited in that the large introns exceeding 1000 bp are fewer in numbers and transposon-free as compared to the large introns among animals,where the larger introns are filled with transposable elements and appear expansion-flexible,reaching several kilobasepairs(kbp)and even thousands of kbp in size.Most of the intron parameters can be studied as signatures of the specific splicing machineries of different eukaryotic lineages and are highly relevant to the regulation of gene expression and functionality.In particular,the transcription-splicing-export coupling of eukaryotic intron dispensing leads to a working hypothesis that all intron parameters are evolved to be efficient and function-related in processing and routing the spliced transcripts.展开更多
文摘CRISPR/Cas9[Clustered regular interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9]系统是广泛存在于细菌和古生菌中可降解入侵病毒和噬菌体DNA的一种基因定点编辑技术,由于其具有快速、精准、高效,易于设计,且特异性高等优势,得到了广泛的应用。概述了CRISPR/Cas9基因编辑技术的发展历程,并介绍其结构和编辑原理、在动物细胞系构建中的应用及展望,以便为更合理地应用和改进该技术提供系统的文献参考。
文摘We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.
基金National Natural Science Foundation of China （#90208011, #30300174, #30070856 , #30421005） National Key Basic Research and Development Program of China （#2002CB713802 , #2005CB522700） Shanghai Key Project of Basic Science Research （#04DZ14005 , #04DZ05608）.
文摘Members of the basic helix-loop-helix （bHLH） gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer （FRET） is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD 1, Mash 1, Neurogenin 1 （Ngn 1）, Neurogenin2 （Ngn2）, and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein （EYFP） and enhanced cyan fluorescence protein （ECFP）, respectively. The subcellular localization and mobility ofbHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mashl/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.
基金The Knowledge Innovation Program of the Chinese Academy of Sciences,(No.KSCX2-EW-G-8)the National Basic Research Program of China program(No.2009CB118903)
文摘As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.
基金The National Natural Science Foundation of China (30670094, 30700028)the Ph.D. Programs Foundation of Ministry of Education of China (2006-0023008)
文摘Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.
文摘中国科学院生态环境研究中心汪海林研究团队在生物分析与表观遗传领域取得新进展。该研究在哺乳动物细胞系中检测到的DNA N^6-甲基腺嘌呤(6mA)并不依赖于甲基化转移酶,并发现了DNA聚合酶依赖的DNA 6mA新来源。相关研究工作以“N^6-methyladenine is incorporated into mammalian genome by DNA polymerase”(N^6-甲基腺嘌呤由DNA聚合酶掺入哺乳动物基因组)为题于2020年4月30日在线发表于Cell Research(https://doi.org/10.1038/s41422-020-0317-6)。
基金supported by the National Natural Science Foundation of China(31101063,31271386)National Basic Research Program of China(2010CB126604,2011CB944100,2011CB944101)
文摘All eukaryotic genomes have genes with introns in variable sizes.As far as spliceosomal introns are concerned,there are at least three basic parameters to stratify introns across diverse eukaryotic taxa:size,number,and sequence context.The number parameter is highly variable in lower eukaryotes,especially among protozoan and fungal species,which ranges from less than4%to 78%of the genes.Over greater evolutionary time scales,the number parameter undoubtedly increases as observed in higher plants and higher vertebrates,reaching greater than 12.5 exons per gene in average among mammalian genomes.The size parameter is more complex,where multiple modes appear at work.Aside from intronless genes,there are three other types of intron-containing genes:half-sized,minimal,and size-expandable introns.The half-sized introns have only been found in a limited number of genomes among protozoan and fungal lineages and the other two types are prevalent in all animal and plant genomes.Among the size-expandable introns,the sizes of plant introns are expansion-limited in that the large introns exceeding 1000 bp are fewer in numbers and transposon-free as compared to the large introns among animals,where the larger introns are filled with transposable elements and appear expansion-flexible,reaching several kilobasepairs(kbp)and even thousands of kbp in size.Most of the intron parameters can be studied as signatures of the specific splicing machineries of different eukaryotic lineages and are highly relevant to the regulation of gene expression and functionality.In particular,the transcription-splicing-export coupling of eukaryotic intron dispensing leads to a working hypothesis that all intron parameters are evolved to be efficient and function-related in processing and routing the spliced transcripts.
