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核酸酶基因LoENDONUCLEASE1在东方百合花朵衰老过程中的功能分析 被引量:1
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作者 李蕊蕊 刘玉杰 +4 位作者 许鑫彤 刘熠 何颖姣 王彩云 罗靖 《华中农业大学学报》 CAS CSCD 北大核心 2024年第1期157-165,共9页
为明确百合(Lilium spp.)花朵衰老发生的分子机制,在东方百合‘西伯利亚’(Lilium oriental hybrid‘Siberia’)花被片中克隆了一个衰老相关基因LoENDONUCLEASE 1(SAG10),利用qRT-PCR进行基因表达分析,并通过农杆菌介导的拟南芥稳定表... 为明确百合(Lilium spp.)花朵衰老发生的分子机制,在东方百合‘西伯利亚’(Lilium oriental hybrid‘Siberia’)花被片中克隆了一个衰老相关基因LoENDONUCLEASE 1(SAG10),利用qRT-PCR进行基因表达分析,并通过农杆菌介导的拟南芥稳定表达系统和百合花被片瞬时表达体系验证LoENDONUCLEASE 1基因功能。结果显示,LoENDONUCLEASE 1基因开放阅读框(ORF)为921 bp,编码306个氨基酸;LoENDONU⁃CLEASE 1在百合花朵和叶片的衰老过程中特异表达,且受到脱落酸和水杨酸的诱导;拟南芥过表达LoENDO⁃NUCLEASE 1株系中叶片叶绿素含量显著低于对照株系,百合花被片中瞬时过表达LoENDONUCLEASE 1基因加速了百合花被片的衰老,且过表达花被片中丙二醛和离子渗透率含量显著高于对照。结果表明,LoEN⁃DONUCLEASE 1具有促进花朵和叶片衰老的功能。 展开更多
关键词 百合 鲜切花 花朵衰老 衰老相关基因 核酸酶 过表达
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mitoTALENs植物线粒体基因编辑载体的构建方法
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作者 周家伟 武志强 《生物技术通报》 CAS CSCD 北大核心 2024年第10期172-180,共9页
【目的】mitoTALENs植物线粒体基因编辑技术能够高效地实现线粒体基因的敲除,进而有效实现线粒体基因功能的研究,但mitoTALENs载体构建过程非常的繁琐复杂且目前仍没有较为系统完整的载体构建方法作为参考。为解决这个问题,结合前人已... 【目的】mitoTALENs植物线粒体基因编辑技术能够高效地实现线粒体基因的敲除,进而有效实现线粒体基因功能的研究,但mitoTALENs载体构建过程非常的繁琐复杂且目前仍没有较为系统完整的载体构建方法作为参考。为解决这个问题,结合前人已发表的及本实验室摸索出的方法对mitoTALENs载体构建的完整过程进行了详尽描述,为之后利用mitoTALENs技术进行植物线粒体基因功能研究的研究者们提供重要参考。【方法】以水稻线粒体WA352基因作为目的基因,利用其序列特异性区域设计了靶点TAL,首先采用Platinum gate TALEN assembly的两步组装技术分别构建了mitoTALENs的TALEN-left和TALEN-right载体,然后利用multisite LR反应将TALEN-left、TALEN-right及含有其他功能元件的进入载体和目的载体进行反应,生成最终的表达载体。【结果】第一步组装构建10个载体,第二步组装构建2个载体,最后通过multisite LR反应构建1个终表达载体。【结论】详细介绍了mitoTALENs载体的构建过程,为该技术使用者提供重要参考,以促进植物线粒体基因编辑研究领域的发展。 展开更多
关键词 线粒体基因编辑 mitotalens载体 Platinum gate talen assembly multisite LR
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Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay(NPA-SH) 被引量:11
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作者 Zhen Yu Mi Tiezhu Yu Zhigang 《Journal of Environmental Sciences》 SCIE EI CAS CSCD 2008年第12期1481-1486,共6页
Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qu... Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c... 展开更多
关键词 harmful algae blooms Phaeocystis globosa Scherffel sandwich hybridization integrated with nuclease protection assay (NPA-SH)
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Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay 被引量:2
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作者 CHEN Jie ZHEN Yu +1 位作者 MI Tiezhu YU Zhigang 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2009年第2期121-126,共6页
Prorocentrum donghaiense is an important harmful algae bloom (HAB) causing creature in China's seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects... Prorocentrum donghaiense is an important harmful algae bloom (HAB) causing creature in China's seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay (NPA-SH) was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x+ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense. 展开更多
关键词 Prorocentrum donghaiense ribosomal RNA S1 enzyme sandwich hybridization integrated with nuclease protection assay (NPA-SH)
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Preparation of human decellularized peripheral nerve allograft using amphoteric detergent and nuclease 被引量:2
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作者 Joo-Yul Bae Suk Young Park +2 位作者 Young Ho Shin Shin Woo Choi Jae Kwang Kim 《Neural Regeneration Research》 SCIE CAS CSCD 2021年第9期1890-1896,共7页
Animal studies have shown that amphoteric detergent and nuclease(DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents... Animal studies have shown that amphoteric detergent and nuclease(DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents(Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review. 展开更多
关键词 median nerve sural nerve nuclease DETERGENT human decellularized nerve graft
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Genome engineering and disease modeling via programmable nucleases for insulin gene therapy;promises of CRISPR/Cas9 technology 被引量:2
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作者 Yunus E Eksi Ahter D Sanlioglu +2 位作者 Bahar Akkaya Bilge Esin Ozturk Salih Sanlioglu 《World Journal of Stem Cells》 SCIE 2021年第6期485-502,共18页
Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases... Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases,zinc finger nucleases,transcription activator-like effector nucleases and RNA-guided engineered nucleases(RGENs),which create double-strand breaks at specific target sites in the genome,and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism.A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype,without the need for the reengineering of the specific enzyme when targeting different sequences.CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function.RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes,as summarized and exemplified in this manuscript. 展开更多
关键词 Programmable nucleases CRISPR/Cas9 Stem cells Disease modeling DIABETES Insulin gene therapy
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Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis
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作者 高光宇 李渝 +3 位作者 王伟 王树峰 Dongping Zhong 龚旗煌 《Chinese Physics B》 SCIE EI CAS CSCD 2015年第1期81-88,共8页
Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectrosc... Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule's cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity. 展开更多
关键词 ultrafast spectroscopy protein dynamics staphylococcal nuclease(SNase) site-directed mutagenesis
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The Distribution and Substrate Specificity of Extracellular Nuclease Activity in Marine Fungi
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作者 Larissa A. Balabanova Michael V. Pivkin Valery A. Rasskazov 《Open Journal of Marine Science》 2012年第4期188-195,共8页
The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium,... The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium, Scopulariopsis, Wardomyces, Periconia, have implied its important function in the organic phosphorus and nitrogen circle in the Ocean. The fungal nucleases of 64 isolates tested were more or less specific for single-stranded DNA with a high preferential specificity towards poly-U substrate with forming of 5’-phosphate mononucleotides. A couple of the nucleases were capable of RNA digesting. The highest level of extracellular nucleolytic ability was observed in Penicillium spp. isolates. The tight correlation found between extracellular nuclease activity and the rate of thymidine uptake by actively growing and sporulating marine fungus Penicillium melinii suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions. 展开更多
关键词 MARINE Fungi MARINE Environment Single-Strand-Specific nuclease DNASE RNAse SSDNA THYMIDINE Uptake
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Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine
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作者 Yong Ma Buyun Chen Donglu Zhang 《Journal of Pharmaceutical Analysis》 SCIE CAS CSCD 2020年第3期247-252,共6页
Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficie... Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time.The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions.Deoxyadenosine monophosphate(dAMP)was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis.The linear range in DNA quantitation by this method is 1.2-5000 ng/mL.In the validation,the inter-day and intra-day accuracies were within 90%-110%,and the inter-day and intra-day precision were acceptable(RSD<10%).The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model.Compared to the quantitation methods using UV absorbance,the reported method provides an enhanced sensitivity,and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide. 展开更多
关键词 nuclease P1 UPLC-MS/MS DNA quantitation DNA alkylation Pyrrolobenzodiazepine(PBD-Dimer)
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Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra
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作者 PARK Mirye PARK So Yun +4 位作者 HWANG Jinik JUNG Seung Won 3LEE Juyun CHANG Man LEE Taek-Kyun 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2018年第5期107-112,共6页
Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquet... Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization(NPA-SH) probe that targets the large subunit of ribosomal RNA(LSU r RNA). We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H.triquetra at a concentration of 1.5×10^4 cells/m L, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015(3.0×10^4 cells/m L). We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H.triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions. 展开更多
关键词 nuclease protection assay sandwich hybridization Heterocapsa triquetra red tide monitoring
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Transcribed single nucleotide polymorphism: Ideal markers for detecting gene imprinting by 5' nuclease assay
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作者 朱冠山 万谟彬 +1 位作者 朱忠政 郑瑞英 《Journal of Medical Colleges of PLA(China)》 CAS 2002年第4期242-246,共5页
Objective: To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the ge... Objective: To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the genotype of a transcribed single nucleotide polymorphism (SNP) rs705(C>T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lym-phoblast cell lines. Results: Allele discrimination showed a clear monoallelic expression pattern of SNRPN, which was confirmed by RT-PCR based restriction fragment length polymorphism (RFLPs). Pedigree analysis verified the paternal origin of expressed allele, which was in consistency with previous report. Conclusion: Transcribed SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach also may be used to discover differential allele expression of non-imprinted genes, finding out gene cis-acting functional polymorphism. 展开更多
关键词 single nucleotide polymorphism genomic imprinting 5' nuclease assay
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Spectroscopic Characterization of Staphylococcal Nuclease Mutants with Tryptophan at Internal Sites
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作者 高光宇 李渝 +3 位作者 王伟 仲冬平 王树峰 龚旗煌 《Chinese Physics Letters》 SCIE CAS CSCD 2015年第4期151-155,共5页
Tryptophan (Trp) is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the int... Tryptophan (Trp) is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type (WT). Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins. 展开更多
关键词 WT Spectroscopic Characterization of Staphylococcal nuclease Mutants with Tryptophan at Internal Sites ANS
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原核Argonaute的应用研究进展
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作者 王飞 李文强 +4 位作者 刘洋 王珑瑜 陈晚苹 崔佳凯 马立新 《华中农业大学学报》 CAS CSCD 北大核心 2024年第4期82-93,共12页
原核生物Argonautes(pAgos)是参与细胞防御外来DNA入侵的可编程核酸酶。在体外,pAgos可以结合小的单链核酸(ssDNA/ssRNA)向导来识别和切割互补DNA/RNA。在体内,pAgos优先靶向多拷贝遗传元件、噬菌体和质粒,从而抑制入侵核酸的扩增和噬... 原核生物Argonautes(pAgos)是参与细胞防御外来DNA入侵的可编程核酸酶。在体外,pAgos可以结合小的单链核酸(ssDNA/ssRNA)向导来识别和切割互补DNA/RNA。在体内,pAgos优先靶向多拷贝遗传元件、噬菌体和质粒,从而抑制入侵核酸的扩增和噬菌体感染。pAgos作为一类新兴的可编程核酸酶,比目前应用最为广泛的CRISPR-Cas系统更具灵活性,在生物技术方面展现出巨大的潜力。早期的研究聚焦于嗜热的pAgo,目前基于嗜热pAgos的主要应用包括分子诊断和体外DNA组装。为了推进基于Ago的体内生物技术,如基因编辑的应用,研究人员的焦点逐渐转移到中温生物来源的pAgos,虽然目前pAgos还未实现基因组编辑,但是随着越来越多的pAgo被发掘以及研究人员对pAgos催化机制的深入研究,有望开发基于pAgos的下一代基因编辑技术。本文总结了已知代表性pAgos和基于pAgos发展的生物技术,并简要分析了pAgos在原核生物和真核生物体内应用面临的挑战和可能的应对策略。 展开更多
关键词 可编程核酸酶 原核Argonaute 基因编辑 分子诊断 DNA组装
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科教融汇理念下基于技术服务的高素质技术技能人才培养实践研究
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作者 龙玲 李秀鑫 +2 位作者 张波 林盛 段治强 《成都航空职业技术学院学报》 2024年第3期4-7,共4页
为了更好地满足产业转型升级背景下企业创新技术发展需求,促进科研与教学的联动性,针对目前高职开展科教融汇载体不清晰、机制不畅通、实施路径不适用等问题,提出高职院校与企业深入合作开展技术服务,构建“项目驱动、任务分解、能力培... 为了更好地满足产业转型升级背景下企业创新技术发展需求,促进科研与教学的联动性,针对目前高职开展科教融汇载体不清晰、机制不畅通、实施路径不适用等问题,提出高职院校与企业深入合作开展技术服务,构建“项目驱动、任务分解、能力培养、岗位胜任”的高素质技术技能人才培养模式,探索以项目研制过程融入人才培养过程的实施路径,培养与项目任务相匹配、与高技能工作岗位相适应的高素质技术技能人才。 展开更多
关键词 技术服务 高职院校 高素质技术技能 人才培养
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类转录激活因子效应物核酸酶(TALEN)介导的基因组定点修饰技术 被引量:52
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作者 沈延 肖安 +3 位作者 黄鹏 王唯晔 朱作言 张博 《遗传》 CAS CSCD 北大核心 2013年第4期395-409,共15页
人工构建的序列特异性核酸内切酶能够识别并切割特定的DNA靶序列,造成双链断裂,从而引起基因组结构的定点改变。人工核酸内切酶技术使得研究人员有可能对任意物种的基因组进行定点修饰,开启了反向遗传学研究的新天地。类转录激活因子效... 人工构建的序列特异性核酸内切酶能够识别并切割特定的DNA靶序列,造成双链断裂,从而引起基因组结构的定点改变。人工核酸内切酶技术使得研究人员有可能对任意物种的基因组进行定点修饰,开启了反向遗传学研究的新天地。类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)自2010年底开始成功应用于基因打靶,很快成为一种比锌指核酸酶(Zinc-finger nuclease,ZFN)更容易设计、特异性更高和毒性更低的人工核酸内切酶。文章综述了TALEN技术的研究进展及应用前景,重点介绍TALEN的结构、作用机制与构建方法和利用TALEN进行基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,特别是在斑马鱼中的应用,为开展这一领域的研究工作提供参考。 展开更多
关键词 类转录激活因子效应物(TALE) 类转录激活因子效应物核酸酶(talen) 人工核酸内切酶(EEN) 基因组编辑 基因组定点修饰
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利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因 被引量:8
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作者 杨翠翠 佟慧丽 +4 位作者 马兴红 杜巍 刘丹 杨宇 严云勤 《遗传》 CAS CSCD 北大核心 2014年第7期685-690,共6页
肌肉生长抑制素(Myostatin,MSTN)基因能够负向调节骨骼肌的生长和发育,牛MSTN基因突变会出现"双肌"特征。文章利用转录激活因子样效应物核酸酶(TALENs)靶向敲除牛胎儿成纤维细胞的MSTN基因,获得敲除MSTN基因的细胞系,为制备M... 肌肉生长抑制素(Myostatin,MSTN)基因能够负向调节骨骼肌的生长和发育,牛MSTN基因突变会出现"双肌"特征。文章利用转录激活因子样效应物核酸酶(TALENs)靶向敲除牛胎儿成纤维细胞的MSTN基因,获得敲除MSTN基因的细胞系,为制备MSTN基因敲除牛提供材料。构建一对MSTN基因的TALENs真核表达载体,分别采用PEI转染试剂和电穿孔法进行牛胎儿成纤维细胞的转染,测序结果表明TALEN技术可用于敲除牛MSTN基因,利用T7核酸内切酶1(T7E1)检测其突变效率,结果显示电穿孔转染的敲除效率为20.4%。通过有限稀释法,共获得10个MSTN基因敲除的细胞克隆(包括MSTN-/-和MSTN+/-),其靶位点敲除的碱基数分别是1~20不等,部分会出现移码突变。出现移码突变的细胞系可用于MSTN基因敲除的转基因肉牛的制备。 展开更多
关键词 talens MYOSTATIN 敲除 转染
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TALEN构建与斑马鱼基因组定点突变的实验方法与流程 被引量:21
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作者 沈延 黄鹏 张博 《遗传》 CAS CSCD 北大核心 2013年第4期533-544,共12页
类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)是最近发展起来的一类新型的人工核酸内切酶,它由特异性的TALE DNA结合结构域和非特异性的FokⅠ核酸内切酶切割结构域组成。TALEN能够根据用户需要... 类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)是最近发展起来的一类新型的人工核酸内切酶,它由特异性的TALE DNA结合结构域和非特异性的FokⅠ核酸内切酶切割结构域组成。TALEN能够根据用户需要切割特定的核苷酸靶序列,造成DNA双链断裂,从而诱导该靶序列产生indel突变,目前已成功地应用于多个物种或体外培养细胞的基因组定点突变。文章介绍TALEN靶点的选择与确认,采用"单元组装法"构建人工TALEN的原理与步骤,以及通过显微注射TALEN mRNA诱导并筛选斑马鱼突变体的实验流程与经验。这些方法理论上也适用于对其他物种进行基因打靶。 展开更多
关键词 类转录激活因子效应物核酸酶(talen) 斑马鱼 基因组定点突变 基因打靶 反向遗传学技术
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应用TALEN技术对兔CCR5基因进行靶向修饰 被引量:6
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作者 唐成程 张全军 +4 位作者 李小平 樊娜娜 杨翌 全龙泉 赖良学 《遗传》 CAS CSCD 北大核心 2014年第4期360-368,共9页
缺少合适的能够被人免疫缺陷病毒1型(Human immunodeficiency virus 1,HIV-1)感染的动物模型是获得性免疫缺陷综合征/艾滋病(Acquired immunedeficiency syndrome,AIDS)疫苗和药物研发的瓶颈.HIV-1能在野生型兔子中形成持续感染,在... 缺少合适的能够被人免疫缺陷病毒1型(Human immunodeficiency virus 1,HIV-1)感染的动物模型是获得性免疫缺陷综合征/艾滋病(Acquired immunedeficiency syndrome,AIDS)疫苗和药物研发的瓶颈.HIV-1能在野生型兔子中形成持续感染,在共表达人CD4和CCR5的兔细胞系中,HIV-1能高效复制,并形成合胞体.若在家兔中高表达人CD4和CCR5,就可能获得研究AIDS的理想动物模型.文章采用高效基因打靶技术—类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN),探讨在家兔CCR5基因位点定点敲入人CD4和CCR5,获得能够感染HIV-1家兔模型的可能性.针对家兔CCR5基因,设计了两对TALENs和一个同源打靶载体,TALEN mRNAs和DNA同源片段显微注射到家兔受精卵中,体外培养3~5 d后,收集24枚胚胎,对胚胎的基因突变情况进行PCR和测序分析.结果显示,在家兔CCR5位点,24枚胚胎均发生了基因敲除,5枚胚胎还发生了人CD4和CCR5基因敲入.该结果为建立艾滋病研究新动物模型奠定了基础. 展开更多
关键词 类转录激活因子效应物核酸酶(talen) 家兔 CCR5基因 基因修饰
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IL2rg^(-/-)大鼠支持人RSV在体内的长期感染
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作者 熊芮 吴勇 +8 位作者 杨艳伟 屈哲 刘甦苏 王誉雅 马丽颖 付瑞 彭宜红 梁春南 范昌发 《中国实验动物学报》 CAS CSCD 北大核心 2024年第1期17-24,共8页
目的为了克服已有人呼吸道合胞病毒(human respiratory syncytial virus,hRSV)动物模型的局限性,如半受纳性和感染持续时间短,本文利用TALEN基因编辑技术建立了IL2rg基因敲除(IL2rg^(-/-))的大鼠模型。方法用hRSV滴鼻感染该动物模型,观... 目的为了克服已有人呼吸道合胞病毒(human respiratory syncytial virus,hRSV)动物模型的局限性,如半受纳性和感染持续时间短,本文利用TALEN基因编辑技术建立了IL2rg基因敲除(IL2rg^(-/-))的大鼠模型。方法用hRSV滴鼻感染该动物模型,观察感染期(0~35 d)的临床表征、体重及体温变化;记录不同时间点(滴鼻感染后第4、11、20、35天)鼻腔、气管、肺等呼吸道脏器的病毒总拷贝数;在观察终点(滴鼻感染后第35天)对感染动物的靶器官进行病理分析;观察不同时间点(滴鼻感染后第4、20、35天)外周血T、B、NK、NKT细胞的变化及不同时间点多种细胞因子的变化。结果(1)通过鼻内接种hRSV后,纯合的IL2rg基因敲除大鼠的呼吸道内能保持较高的病毒载量,鼻腔中病毒的平均峰值滴度能快速升至1×10^(10 )copies/g,至第5周时,病毒依然能维持复制,病毒载量亦可达到1×10^(7)copies/g。(2)但其鼻、气管和肺组织,无明显病变。(3)感染hRSV的IL2rg^(-/-)大鼠外周血B细胞含量有上升。(4)IL-6和MCP-1两种细胞因子都在感染前期上升,在观察终点回落。结论本研究基于TALEN技术建立了IL2rg^(-/-)大鼠模型,并发现该模型能很好地支持hRSV高水平复制和并长期感染,为抗病毒药物筛选、抗hRSV抗体体内效力评价,提供了良好的动物模型。 展开更多
关键词 IL2rg-/-大鼠 talen基因编辑技术 NK细胞缺陷 人呼吸道合胞病毒 感染动物模型
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基因编辑技术及其在畜禽遗传育种中的应用研究进展
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作者 曹慧 韩博 孙东晓 《中国畜牧杂志》 CAS CSCD 北大核心 2024年第7期6-12,共7页
基因编辑是指对生物体基因组特定的DNA进行改造,使生物的性状发生定向的、可遗传的改变。基因编辑技术主要包括锌指核酸酶(Zinc Finger Nucleases,ZFNs)技术、转录激活因子样效应物核酸酶(Transcription Activator-Like Effector Nuclea... 基因编辑是指对生物体基因组特定的DNA进行改造,使生物的性状发生定向的、可遗传的改变。基因编辑技术主要包括锌指核酸酶(Zinc Finger Nucleases,ZFNs)技术、转录激活因子样效应物核酸酶(Transcription Activator-Like Effector Nucleases,TALENs)技术、成簇规则间隔短回文重复序列/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated Protein 9,CRISPRs/Cas9)技术。在畜禽中使用高效且精确的基因编辑技术可以提高畜禽产量、品质、抗病力等。本文从基因编辑技术的发展、原理及其在畜禽遗传育种中的应用进行综述,为基因编辑技术应用于畜禽遗传育种的研究提供参考。 展开更多
关键词 基因编辑 畜禽 ZFNs talens CRISPR/Cas9
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