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Sustained high level transgene expression in mammalian cells mediated by the optimized piggybac transposon system

Genes & Diseases 2015年第1期2卷 96-105页

作者： Xiang Chen Jing Cui Zhengjian Yan Hongmei Zhang Xian Chen Ning Wang Palak Shah Fang Deng Chen Zhao Nisha Geng Melissa Li Sahitya K.Denduluri Rex C.Haydon Hue H.Luu Russell R.Reid Tong-Chuan He Molecular Oncology Laboratory Department of Orthopaedic Surgery and Rehabilitation MedicineThe University of Chicago Medical CenterChicagoILUSA Department of Pediatric Oncology Baylor College of MedicineHoustonTXUSA Ministry of Education Key Laboratory of Diagnostic Medicine and The Affiliated Hospitals of Chongqing Medical UniversityChongqingChina Department of Laboratory Medicine The Affiliated Hospitals of Qingdao UniversityQingdaoChina Departments of Oncology Cell Biology and Laboratory MedicineThird Military Medical UniversityChongqingChina Section of Plastic&Reconstructive Surgery Department of SurgeryThe University of Chicago Medical CenterChicagoILUSA

Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene ***,stable transgene expression has been accomplished by using retroviral or lentiviral ***,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling *** piggybac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian *** its inherent advantages over lentiviral and retroviral systems,piggybac system has not been widely used,at least in part due to their limited manipulation ***,we seek to optimize piggybac-mediated transgene expression and generate a more efficient,user-friendly piggybac *** engineering a panel of versatile piggybac vectors and constructing recombinant adenoviruses expressing piggybac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase *** further determine the drug selection timeline to achieve optimal stable transgene ***,we demonstrate that the transgene copy number of piggybac-mediated integration is approximately 10 times higher than that mediated by retroviral *** the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high ***,these results strongly suggest that the optimized piggybac system is a valuable tool for making stable cell lines with sustained,high transgene expression.

关键词： Mesenchymal stem cells piggybac transposon piggybac transposase Retroviral vectors Stable transgene expression Transposition

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piggybac transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

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Journal of Northeast Agricultural University(English Edition) 2012年第2期19卷 33-41页

作者： Luo Yi-bo Zhang Li Zhu Jiang Wu Mei-ling Huan Yan-jun Yin Zhi Mu Yan-shuang Xia Ping LiuZhong-hua College of Life Sciences Northeast Agricultural University Harbin 150030 China University of California at Davis Sacramento California USA

piggybac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggybac transposon marked with enhanced green fluorescent protein （eGFP） and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggybac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. piggybac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggybac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer （SCNT）, the cleavage rate and blastocyst rate of constructed embryos harboring piggybac transposition had no difference with random insertion group. This study provides key information on the piggybac transposon system as a tool for creating transgenic pigs.

关键词： piggybac transposon eGFP somatic cell nuclear transfer pig transgene

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Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

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World Journal of Gastroenterology 2012年第47期18卷 6935-6942页

作者： Kazuya Shinmura Masanori Goto Hong Tao Shun Matsuura Tomonari Matsuda Haruhiko Sugimura Department of Tumor Pathology Hamamatsu University School of MedicineShizuoka 431-3192Japan Research Center for Environmental Quality Management Kyoto UniversityShiga 520-0811Japan

AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human ***:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this *** H1299 cancer cell lines inducibly expressing wild-type(WT) MUTYH(type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggybac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH *** expression was examined after cumate induction using Western blotting analysis and immunofluorescence *** intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently ***,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation ***:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with *** of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged *** mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of *** mutation frequency(4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells(P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55 × 10-2

关键词： 8-hydroxyguanine Mutation MUTYH MUTYH-associated polyposis Oxidative mutagenesis supF forward mutation assay piggybac transposon Colorectal polyposis

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Robust genome and RNA editing via CRISPR nucleases in piggybac systems

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Bioactive Materials 2022年第8期7卷 313-320页

作者： Yuqian Jiang Rachel Catherine Hoenisch Yun Chang Xiaoping Bao Craig E.Cameron Xiaojun Lance Lian Department of Biomedical Engineering Pennsylvania State UniversityUniversity ParkPA16802USA Huck Institutes of the Life Sciences Pennsylvania State UniversityUniversity ParkPA16802USA Department of Biology Pennsylvania State UniversityUniversity ParkPA16802USA Davidson School of Chemical Engineering Purdue UniversityWest LafayetteIN47907USA Department of Microbiology and Immunology University of North Carolina School of MedicineChapel HillNC27599USA

CRISPR/Cas-mediated genome editing in human pluripotent stem cells(hPSCs)offers unprecedented opportunities for developing in vitro disease modeling,drug screening and cell-based *** efficiently deliver the CRISPR components,here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference *** vectors utilized the piggybac transposon system,which allows stable expression of CRISPR components in *** Cas9 vector PB-CRISPR exhibited high efficiency(up to 99%)of inducing gene knockout in both protein-coding genes and long non-coding *** other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown(98%knockdown for CD90)with optimized gRNA *** together,our piggybac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.

关键词： CRISPR-Cas9 Genome editing Cas13d RNA editing piggybac transposon Human pluripotent stem cells

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Expression of the B Subunit of Escherichia coli Heat-labile Enterotoxin in Transformed Bombyx mori BmN Cells

Expression of the B Subunit of Escherichia coli Heat-labile ...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Wen-Lin.Zhou Jin-Ru Cao Ai-Hong Ye Cheng-Liang Gong Hong-Biao Weng Li-Hua He Ren-Yu Xue Guang-Li Cao Yong-Qiang Wang Sericulture Research Institute Zhejiang Academy of Agricultural Sciences College of Pre-clinical Medical and Biological Science Soochow University National Engineering Laboratory for Modern Silk

The non-toxic B subunit of Escherichia coli heat-labile enterotoxin(LTB)is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. To obtain transformed silkworm cell line stably expressing LTB, we fused the LTB coding sequence, neomycin- resistance gene(NeoR), and gfp gene into piggy Bac-based transponson vector, and transduced into silkworm BmN cells. After screening against antibiotic G418, The positive rate of cells emitting green fluorescence was 70.79%. PCR detection indicates the existence of exogenous LTB coding sequence, NeoR, and gfp in the transformed cell genome. Western blot analysis also confirms the predicated 0 kDa band of LTB protein. These results demonstrated that our strategy was practicable.

关键词： Escherichia coli Heat-labile Enterotoxin piggybac transposon Bombyx mori BmN cell Genetic transformation

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Germline Transformation of the Silkworm Bombyx mori L. by Sperm-Mediated Gene Transfer

Germline Transformation of the Silkworm Bombyx mori L. by Sp...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Wenlin Zhou Chengliang Gong Renyu Xue Guangli Cao Jinru Cao Aihong Ye HongbiaoWeng Yongqiang Wang Sericulture Research Institute Zhejiang Academy of Agricultural Sciences Preclinical Medical and Biological Science College Soochow University National Engineering Laboratory for Modern Silk

The domesticated silkworm Bombyx mori L. has important roles in basic biological research and applied science. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycin-resistance gene（NeoR）and the green fluorescent protein gene（gfp）into the piggy Bac-based transposon vector and transduced it into silkworms by sperm-mediated gene transfer（SMGT）. Fluorescence observation indicated the positive rate of G0 egg-batches is 72.7%. After screening against the antibiotic G418, development of individual larvae in the same brood showed significant size differences. PCR detection indicated the existence of gfp and Neo R and confirmed the positive rate of transgenesis as 0.47%. Southern blot analysis confirmed the presence of the exogenous genes in the genome of G7 larvae. These results show that our strategy is practical and markedly improves the efficiency of SMGT.

关键词： Bombyx mori L. piggybac transposon sperm-mediated gene transfer transgenesis

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