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piggyBac as a high-capacity transgenesis and genetherapy vector in human cells and mice

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piggyBac as a high-capacity transgenesis and genetherapy vec...

Preliminary Study on transgenesis by Injecting Exogenous DNA into Zygote Cytoplasm of Buffalo

中国遗传学会第九次全国会员代表大会暨学术研讨会

作者：李荣波 ZHUANG Yuan HAN Min XU Tian 吴晓晖 State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine Fudan-Yale Biomedical Research CenterSchool of Life SciencesFudan University

The stable genomic integration and expression of a large transgene is a major hurdle in gene *** show that the modified piggyBac(PB) transposon system can be used to introduce a 207 kb genomic DNA fragment containing the RORγ/γt locus into human cells and ***-mediated transgenesis results in a single copy of a stably inherited and expressed *** results indicate that PB could serve as an effective highcapacity vector for functional analysis of the mammalian genome and for gene therapy in human cells.

关键词： piggyback transgenesis Gene therapy

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Agricultural Science & Technology 2012年第6期13卷 1167-1170,1174页

作者：陈自洪崔奎青孟凡丽刘玉兵王丹陆凤花石德顺广西大学亚热带生物资源保护利用国家重点实验室广西南宁530004 广西壮族自治区人口和计划生育研究中心广西南宁530021 山东潍坊市人民医院生殖医学中心山东潍坊261100

[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation. One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization （IVF）; the other group of oocytes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection （generally called ICSI-Mediated Gene Transfer, ICSI-Tr）. Expression of exogenous DNA was observed and recorded during the process of embryonic development. [Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF injection group showed no significant difference compared with that in ICSI-Tr group （P0.05）. In addition, the cleavage rate and early embryonic gene expression efficiency in IVF injection group were significantly higher with injection at 7-10 h post IVF than that at 18-20 h post IVF （P0.05）. [Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes, and the optimal injection time is 7-10 h post IVF.

关键词： Buffalo Zygote Cytoplasmic injection transgenesis

Biotechnological interventions for the sustainable management of a global pest,whitefly(Bemisia tabaci)

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Insect Science 2021年第5期28卷 1228-1252页

作者： Archna Suhag Honey Yadav Darshna Chaudhary S.Subramanian Ranjana Jaiwal Pawan K.Jaiwal Department of Zoology M.D.UniversityRohtakIndia Centre for Biotechnology M.D.UniversityRohtakIndia Division of Entomology Indian Agriculture Research InstituteNew DelhiIndia

Whiteflies(Bemisia tabaci)are polyphagous invasive hemipteran insects that cause serious losses of important crops by directly feeding on phloem sap and transmitting pathogenic *** insects have emerged as a major threat to global agriculture and food *** synthesized insecticides are currently the only option to control whiteflies,but the ability of whiteflies to evolve resistance against insecticides has made the management of these insects very *** host-plant resistance against whiteflies identified in some crop plants has not been exploited to a great *** engineering approaches,such as transgenics and RNA interference(RNAi),are potentially useful for the control of *** plants harboring insecticidal toxins/lectins developed via nuclear or chloroplast transformation are a promising vehicle for whitefly ***-stranded RNAs(dsRNAs)of several insect genes,delivered either through microinjection into the insect body cavity or orally via an artificial diet and transiently or stably expressed in transgenic plants,have controlled whiteflies in model plants and in some crops at the laboratory level,but not at the field *** this review,we highlight the merits and demerits of each delivery method along with strategies for sustained delivery of dsRNAs via fungal entomopathogen/endosymbiont or nontransgenic RNAi approaches,foliar sprays,root absorption or nanocarriers as well as the factors affecting efficient RNAi and their biosafety *** sequencing and transcriptome studies of whitefly species are facilitating the selection of appropriate genes for RNAi and gene-editing technology for the efficient and resilient management of whiteflies and their transmitted viruses.

关键词： Bemisia tabaci.,gene editing management nanotechnology RNAi transgenesis

Establishment of highly efficient transgenic system for black soldier fly(Hermetia illucens)

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Insect Science 2023年第4期30卷 888-900页

作者： Zongqing Kou Xingyu Luo Yuguo Jiang Bihui Chen Yu Song Yaohui Wang Jun Xui Jeffery K.Tomberlin Yongping Huang Key Laboratory of Insect Developmental and Evolutionary Biology Center for Excellence in Molecular Plant SciencesShanghai Insttute of Plant Physiology and EcologyChinese Academy of SciencesShanghaiChina University of Chinese Academy of Sciences BeijingChina Department of Entomology Texas A&M UniversityCollege StationTXUSA

The black soldier fly(BSF),Hermetia illucens,is a promising insect for miti-gating solid waste problems as its larvae are able to bioconvert organic waste into valuable *** recently reported a high-quality genome assembly of the BSF;analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of *** enable genetic manip-ulation of the BSF,we have established the first transgenic methods for this economically important *** cloned and identified the ubiquitous actin5C promoter(Hiactin5C-p3k)and 3 endogenous U6 promoters(HiU6:1,HiU6:2,and HiU6:3).The Hiactin5C pro-moter was used to drive expression of a hyperactive variant of the piggyBac transposase,which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type ***,we evaluated the 3 HiU6 promoters using this trans-genic ***6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)***,our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.

关键词： actin5C promoter black soldier fly hyPBase transgenesis U6 promoter

Functional genomics studies on the innate immunity of disease vectors

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Insect Science 2008年第1期15卷 15-27页

作者： Luke A. Baton Lindsey Garver Zhiyong Xi George Dimopoulos W. Harry Feinstone Department of Molecular Microbiology and Immunology Malaria Research Institute Bloomberg School of Pubfic Health Johns Hopkins University Baltimore Maryland USA

The increasing availability of genome sequences and the development of highthroughput techniques for gene expression profiling and functional characterization are transforming the study of innate immunity and other areas of insect biology. Already, functional genomic approaches have enabled a quantum advance in the characterization of mosquito immune responses to malaria parasite infection, and similar high-throughput functional genomic studies of other vector-pathogen interactions can be expected in the near future. The application of microarray-based and other expression analyses provide genomewide transcriptional profiles that can be used to identify insect immune system components that are differentially regulated upon exposure to various classes of pathogens, including many important etiologic agents of human and animal diseases. The role of infection-responsive or other candidate immune genes identified through comparative genomic approaches can then be functionally characterized, either in vivo, for instance in adult mosquitoes, or in vitro using cell lines. In most insect vectors of human pathogens, germ-line transgenesis is still technically difficult and maintenance of multiple transgenic lines logistically demanding. Consequently, transient RNA interference （RNAi）-mediated gene-silencing has rapidly become the method of choice for functional characterization of candidate innate immune genes. The powerful combination of transcriptional profiling in conjunction with assays using RNAi to determine gene function, and identify regulatory pathways, together with downstream cell biological approaches to determine protein localization and interactions, will continue to provide novel insights into the role of insect innate immunity in a variety of vector-pathogen interactions. Here we review advances in functional genomics studies of innate immunity in the insect disease vectors, over the past decade, with a particular focus on the Anopheles mosquito and its respo

关键词： Anopheles genomics innate immunity insect mosquito malaria microarray,parasite Plasmodium RNA interference transcriptomics transgenesis vector

Genome editing in Drosophila melanogaster： from basic genome engineering to the multipurpose CRISPR-Cas9 system

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Science China(Life Sciences) 2017年第5期60卷 476-489页

作者： Xingjie Ren Kristof Holsteens Haiyi Li Jin Sun Yifan Zhang Lu-Ping Liu Qingfei Liu Jian-Quan Ni Gene Regulatory Lab School of Medicine Tsinghua University Beijing 100084 China French International School of Hong Kong Hong Kong SAR 999000 China Department of Biology University of California San Diego 92093 USA School of Pharmaceutical Sciences Tsinghua University Beijing 100084 China

Nowadays, genome editing tools are indispensable for studying gene function in order to increase our knowledge of biochemical processes and disease mechanisms. The extensive availability of mutagenesis and transgenesis tools make Drosophila melanogaster an excellent model organism for geneticists. Early mutagenesis tools relied on chemical or physical methods,ethyl methane sulfonate(EMS) and X-rays respectively, to randomly alter DNA at a nucleotide or chromosomal level. Since the discovery of transposable elements and the availability of the complete fly genome, specific genome editing tools, such as P-elements, zinc-finger nucleases(ZFNs) and transcription activator-like effector nucleases(TALENs), have undergone rapid development. Currently, one of the leading and most effective contemporary tools is the CRISPR-cas9 system made popular because of its low cost, effectiveness, specificity and simplicity of use. This review briefly addresses the most commonly used mutagenesis and transgenesis tools in Drosophila, followed by an in-depth review of the multipurpose CRISPR-Cas9 system and its current applications.

关键词： genome editing Drosophila melanogaster mutagenesis transgenesis CRISPR-Cas9

Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2010年第9期11卷 728-734页

作者： Na WANG Cai-ying JIANG Ming-xing JIANG Chuan-xi ZHANG Jia-an CHENG Institute of lnsect Sciences Zhejiang University Hangzhou 310029 China Institute of Biochemistry Zhejiang Sci-Tech University Hangzhou 310018 China

The piggyBac transposon has been long used to integrate foreign DNA into insect ***,undesirable transgene expression can result from random insertions into the *** this study,the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2(S2) *** Gal4-piggyBac transposase has a Gal4 DNA-binding domain(DBD),and the target plasmid has upstream activating sequences(UAS) to which the Gal4 DBD can bind with high *** results indicate that,in the Bm-12 and S2 cells,transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times,respectively,compared to controls,where Gal4-UAS interaction was ***,the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid,although the target-directing specificity was not as high as *** chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in ***.

关键词： Bombyx mori piggyBac Gal4-upstream activating sequences(UAS) Transposition assay transgenesis

Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

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Journal of Oceanology and Limnology 2019年第2期37卷 651-656页

作者： PANG Yunlong LI Yan LIU Zhengyi CUI Yulin QIN Song Key Laboratory of Coastal Biology and Biological Resource Utilization Yantai Institute of Coastal Zone Research Chinese Academy of Sciences Yantai 264003 China University of Chinese Academy of Sciences Beijing 101408 China Yantai Ocean Environmental Monitoring Central Station of State Oceanic Administration Yantai 264006 China

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S . horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S . horneri with analysis of molecular markers. The S . horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein (GFP) is an eff ective transgene reporter for S . horneri and that particle bombardment is a suitable method for transformation of S . horneri . Through selection of four diff erent promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S . horneri .

关键词： green fluorescent protein (GFP) particle bombardment Sargassum horneri transgenesis

Development of genome engineering technologies in cattle: from random to specific

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Journal of Animal Science and Biotechnology 2018年第2期9卷 285-293页

作者： Soo-Young Yum Ki-Young Youn Woo-Jae Choi Goo Jang Department of Veterinary Clinical Science College of Veterinary Medicine and the Research Institute of Veterinary Science Seoul National University Farm Animal Clinical Training and Research Center Institute of Green Bio Science Technology Seoul National University Emergence Center for Food-Medicine Personalized Therapy System Advanced Institutes of Convergence Technology Seoul National University College of Veterinary Medicine Seoul National University

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the ***, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.

关键词： Cattle CRISPR-Cas9 Genome engineering technologies transgenesis Transposon