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Advances in Drosophila gene targeting and related techniques

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Frontiers in Biology 2010年第3期5卷 238-245页

作者： Zhongsheng YU Renjie JIAO State Key Laboratory of Brain and Cognitive Science Institute of BiophysicsChinese Academy of SciencesBeijing 100101China Graduate School of Chinese Academy of Sciences Beijing 100080China

Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes.In Drosophila,compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest.However,applications of gene targeting have been limited because the targeting efficiency varies with different genes,and the time and labor of targeting procedure are intensive.Nevertheless,improvement of gene targeting and development of its variant technologies have received much attention of scientists.Here we review recent progress that has been made in expanding the applications of gene targeting,which include the ϕC31 integration system and zinc-finger nucleases induced gene targeting,and new strategies that generate more efficient and reliable gene targeting.

关键词： gene targeting ends-in ends-out ϕC31 integration system zinc-finger nucleases(ZFNs) homologous recombination(HR) Drosophila melanogaster

gene targeting in Marine Embryonic Stem Cells, a New Method to Manipulate the Mouse Genome

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Progress in Natural Science:Materials International 1994年第1期 22-33页

作者：邓初夏 200 Longwood Avenue Boston Dupartment of genetics Harvard Medical School MA 02115 USA

In eukaryotic cells, homologous recombination between DNA sequences residing in the chromosome and newly introduced DNA is commonly referred to as gene targeting.This emerging technology has proven to be a powerful means for precisely manipulating the mammalian genome. gene targeting in totipotent murine embryonic stem (ES) cells can, theoretically, be used to generate mice of any desired genotype, thus aiding in the study of gene structure and function, as well as mammalian developmental biology. In addition, gene targeting also provides a

关键词： gene targeting ES cells mouse mutants isogenic DNA

Application of Artificially Induced Double-strand Breaks (DSB) and Triplex-forming Oligonucleotides (TFO) in the Improvement of gene targeting Efficiency

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Agricultural Biotechnology 2013年第1期2卷 1-6,12页

作者： Hegang LI Wenke CHENG Ke JIANG Xiaoli REN Yongping JIANG Lele HOU Xiaojing HAO Jinshan ZHAO Qingdao Institute of Animal Science and Veterinary Medicine Animal Husbandry Technology Extension Station of Hainan Province Laixi Husbandry and Veterinary Bureau of Qingdao City Rizhuang Agricultural Service Center of Laixi City

gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.

关键词： gene targeting Double-strand breaks Zinc finger nuclease Homing endonuclease Triplex-forming oligonucleotides

Inhibiting effects on HeLa cell growth by HER-2 gene targeting

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Progress in Natural Science:Materials International 1999年第6期 33-40页

作者：王向东姜琨任庆虎陈培利汪维童坦君高建刚 Academy of Sciences Beijing 100083 Beijing Medical University China Chinese Department of Biochemistry & Molecular Biology institute of Developmental Biology

A plasmid (promoter pPNT 1305) for HER-2 gene targeting was constructed and introduced me HeLa cells by electroporation . After positive selection by G418 and negative selection by Gancyclovir, the targeted cell line (HER-2-1 cells) containing only one copy of HER-2 allies was selected. Northern blot assay showed that the mR-NA expression of HER-2 was significantly less than that of its parental cells. The results of Western blot also showed that HER-2 protein of HER-2 - cells was obviously less than that of HeLa cells. The dala of FA.CS assay demenstrated that after one of the two HER-2 copies in HeLa cells was knocked out, the G1 phase percentage of HER-2 cells was more and the M plus G2 phase percentage of HER-2 -1 cells was less than those of HeLa ceils respectively These reaults show that gene targeting of HER-2 has exhibited the inhibiting effects on the proliferational capability of HeLa cells

关键词： gene targeting HER-2 proto-oncogene.

gene targeting using Agrobacterium tumefaciens-mediated CRISPR-Cas system in crop plants

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Gene targeting using Agrobacterium tumefaciens-mediated CRIS...

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

生态文明建设中的植物学：现在与未来——中国植物学会第十...

作者： Rong-Fang XU Hao LI Rui-Ying QIN Lu WANG Li LI Peng-Cheng WEI Jian-Bo YANG Key Laboratory of Rice genetics Breeding of Anhui Province Rice Research InstituteAnhui Academy of Agricultural Sciences

The type Ⅱ clustered,regularly interspaced,short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9) system is a novel molecular tool of site-specific genome modification.Very recently,the CRISPR-Cas9 system was reported to be successfully performed by transient or biolistic-mediated stable transformation in Arabidopsis,Nicotiana benthamiana and rice.Here,we reported the gene targeting and genome editing in rice via Agrobacterium tumefaciens-mediated CRISPR-Cas system.Three 20nt guide RNA were designed to pair with the distinct sites followed by the protospacer adjacent motif(PAM) of a rice herbicide-resistance gene BEL.After integrating the sgRNA and Cas9 cassettes in one binary vector,the transgenic rice plants

关键词： gene targeting CRISPR/Cas9 Rice Agrobacterium Stable transformation

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World Journal of Clinical Cases 2022年第18期10卷 5934-5945页

作者： Yue-Ying Huang Xiao-Yu Zhang Ping Zhu Ling Ji School of Medical Laboratory Weifang Medical UniversityWeifang 261053Shandong ProvinceChina Department of Laboratory Medicine Peking University Shenzhen HospitalShenzhen 518035Guangdong ProvinceChina

The clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPRassociated(Cas)proteins constitute the innate adaptive immune system in several bacteria and archaea.This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity.Owing to the numerous advantages such as ease of use,low cost,high efficiency,good accuracy,and a diverse range of applications,the CRISPR-Cas system has become the most widely used genome editing technology.Hence,the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine.This study reviews the recently reported application platforms for screening,diagnosis,and treatment of different diseases based on CRISPR/Cas systems.The limitations,current challenges,and future prospectus are summarized;this article would be a valuable reference for future genome-editing practices.

关键词： CRISPR-Cas gene editing Molecular diagnostics gene targeting

gene editing in plants:progress and challenges

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National Science Review 2019年第3期6卷 421-437页

作者： Yanfei Mao Jose Ramon Botella Yaoguang Liu Jian-Kang Zhu Shanghai Center for Plant Stress Biology CAS Center of Excellence in Molecular Plant Sciences Chinese Academy of Sciences School of Agriculture and Food Sciences University of Queensland State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources College of Life Sciences South China Agricultural University Department of Horticulture and Landscape Architecture Purdue University

The clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein 9(Cas9)genome editing system is a powerful tool for targeted gene modifications in a wide range of species,including plants. Over the last few years, this system has revolutionized the way scientists perform genetic studies and crop breeding, due to its simplicity, flexibility, consistency and high efficiency. Considerable progress has been made in optimizing CRISPR/Cas9 systems in plants, particularly for targeted gene mutagenesis. However, there are still a number of important challenges ahead, including methods for the efficient delivery of CRISPR and other editing tools to most plants, and more effective strategies for sequence knock-ins and replacements. We provide our viewpoint on the goals, potential concerns and future challenges for the development and application of plant genome editing tools.

关键词： CRISPR Cas9 genome editing base editing gene targeting crop breeding

A Human t-PA Mutant cDNA Cassette Knocked in the Murine fgfr-4 Locus targeting for Mammary Gland Expression

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Acta Biochimica et Biophysica Sinica 2004年第7期36卷 450-456页

作者： Fu-Yu LIN#, Hong-Xing CHEN#, Xuan CHENG, Xiao YANG*, Ji-Xian DENG*, and Pei-Tang HUANG*Beijing Institute of Biotechnology, No.20 Dongdajie Street, Fengtai District, Beijing 100071, China Beijing Institute of Biotechnology No.20 Dongdajie StreetFengtai District Beijing 100071 China

The expression of foreign gene in transgenic animals produced by pronuclear microinjection is often confounded by the position effects caused by not only the nature of chromosomal integration site but also the number and arrangement of multiple transgene copies. gene targeting provides a new way to overcome these inhibitions by introducing single-copy transgene into a chosen site. The choice of a good chromosomal site will favor transgene expression in a predictable fashion. In this study, we tested a new site (fgfr-4) for foreign gene integration and expression. A t-PA mutant (t-PAm) expression cassette under bovine αs1-casein regulatory sequences was efficiently knocked-in fgfr-4 site through homologous recombination. The t-PAm was expressed in the milk of all targeted mice. Our experiment indicates that the fgfr-4 may be a candidate site for knocking foreign gene to make transgenic animals.

关键词： gene targeting fgfr-4 transgenic mammary gland bioreactor integration site t-PA mutant

Targeted Genome Editing by Recombinant Adeno-Associated Virus(rAAV) Vectors for generating genetically Modified Pigs

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Journal of genetics and Genomics 2012年第6期39卷 269-274页

作者： Yonglun Luo Emil Kofod-Olsen Rikke Christensen Charlotte Brandt Sorensen Lars Bolund Department of Biomedicine Aarhus University Wilhelm Meyers AUe 4 DK-8000 Aarhus C Denmark BGI/HuaDa Jigin-Shenzhen Shenzhen 510813 China

Recombinant adeno-associated virus （rAAV） vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applica- bility to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer （SCNT）, this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subse- quently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

关键词： rAAV gene targeting Homologous recombination SCNT genetically modified pigs

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