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https://www.readbyqxmd.com/read/27910942/improved-bi-allelic-modification-of-a-transcriptionally-silent-locus-in-patient-derived-ipsc-by-cas9-nickase
#1
Reto Eggenschwiler, Mohsen Moslem, Mariane Serra Fráguas, Melanie Galla, Oliver Papp, Maximilian Naujock, Ines Fonfara, Ingrid Gensch, Annabell Wähner, Abbas Beh-Pajooh, Claudio Mussolino, Marcel Tauscher, Doris Steinemann, Florian Wegner, Susanne Petri, Axel Schambach, Emmanuelle Charpentier, Toni Cathomen, Tobias Cantz
Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella...
December 2, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27910041/efficient-genome-editing-in-induced-pluripotent-stem-cells-with-engineered-nucleases-in-vitro
#2
Vittavat Termglinchan, Timon Seeger, Caressa Chen, Joseph C Wu, Ioannis Karakikes
Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27907214/derivation-of-induced-trophoblast-cell-lines-in-cattle-by-doxycycline-inducible-piggybac-vectors
#3
Takamasa Kawaguchi, Dooseon Cho, Masafumi Hayashi, Tomoyuki Tsukiyama, Koji Kimura, Shuichi Matsuyama, Naojiro Minami, Masayasu Yamada, Hiroshi Imai
Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique...
2016: PloS One
https://www.readbyqxmd.com/read/27900536/t%C3%AE-4-overexpression-based-on-the-piggybac-transposon-system-in-cashmere-goats-alters-hair-fiber-characteristics
#4
Bingbo Shi, Qiang Ding, Xiaolin He, Haijing Zhu, Yiyuan Niu, Bei Cai, Jiao Cai, Anming Lei, Danju Kang, Hailong Yan, Baohua Ma, Xiaolong Wang, Lei Qu, Yulin Chen
Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter...
November 29, 2016: Transgenic Research
https://www.readbyqxmd.com/read/27879215/a-human-ve-cadherin-tdtomato-and-cd43-green-fluorescent-protein-dual-reporter-cell-line-for-study-endothelial-to-hematopoietic-transition
#5
Ho Sun Jung, Gene Uenishi, Akhilesh Kumar, Mi Ae Park, Matt Raymond, Dustin Fink, Ethan McLeod, Igor Slukvin
Human embryonic stem cell line WA01 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC+ endothelial cells express tdTomato and CD43+ hematopoietic progenitors express eGFP...
September 2016: Stem Cell Research
https://www.readbyqxmd.com/read/27830190/phenotypic-screens-identify-parasite-genetic-factors-associated-with-malarial-fever-response-in-plasmodium-falciparum-piggybac-mutants
#6
Phaedra Thomas, Jennifer Sedillo, Jenna Oberstaller, Suzanne Li, Min Zhang, Naresh Singh, Chengqi C Q Wang, Kenneth Udenze, Rays H Y Jiang, John H Adams
Malaria remains one of the most devastating parasitic diseases worldwide, with 90% of the malaria deaths in Africa in 2013 attributable to Plasmodium falciparum. The clinical symptoms of malaria include cycles of fever, corresponding to parasite rupture from red blood cells every 48 h. Parasite pathways involved in the parasite's ability to survive the host fever response, and indeed, the functions of ~40% of P. falciparum genes as a whole, are still largely unknown. Here, we evaluated the potential of scalable forward-genetic screening methods to identify genes involved in the host fever response...
September 2016: MSphere
https://www.readbyqxmd.com/read/27827797/the-clustered-regularly-interspaced-short-palindromic-repeats-associated-proteins-system-for-the-induction-of-gene-mutations-and-phenotypic-changes-in-bombyx-mori
#7
Jia Song, Jiaqian Che, Zhengying You, Xiaogang Ye, Jisheng Li, Lupeng Ye, Yuyu Zhang, Qiujie Qian, Boxiong Zhong
To probe the general phenomena of gene mutations, Bombyx mori, the lepidopterous model organism, was chosen as the experimental model. To easily detect phenotypic variations, the piggyBac system was utilized to introduce two marker genes into the silkworm, and 23.4% transposition efficiency aided in easily breeding a new strain for the entire experiment. Then, the clustered regularly interspaced short palindromic repeats/an associated protein (Cas9) system was utilized. The results showed that the Cas9 system can induce efficient gene mutations and the base changes could be detected since the G0 individuals in B...
December 2016: Acta Biochimica et Biophysica Sinica
https://www.readbyqxmd.com/read/27823982/angptl8-reverses-established-adriamycin-cardiomyopathy-by-stimulating-adult-cardiac-progenitor-cells
#8
Shuyuan Chen, Jiaxi Chen, Xing-Li Meng, Jin-Song Shen, Jing Huang, Pintong Huang, Zhaoxia Pu, Nathan H McNeill, Paul A Grayburn
Established adriamycin cardiomyopathy is a lethal disease. When congestive heart failure develops, mortality is approximately 50% in a year. It has been known that ANGPTLs has various functions in lipid metabolism, inflammation, cancer cell invasion, hematopoietic stem activity and diabetes. We hypothesized that ANGPTL8 is capable of maintaining heart function by stimulating adult cardiac progenitor cells to initiate myocardial regeneration. We employed UTMD to deliver piggybac transposon plasmids with the human ANGPTL8 gene to the liver of rats with adriamycin cardiomyopathy...
November 3, 2016: Oncotarget
https://www.readbyqxmd.com/read/27764912/efficient-transgene-expression-system-using-a-cumate-inducible-promoter-and-cre-loxp-recombination-in-avian-cells
#9
Tae Sub Park, Si Won Kim, Jeong Hyo Lee
Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells...
October 20, 2016: Asian-Australasian Journal of Animal Sciences
https://www.readbyqxmd.com/read/27729044/retroviral-vectors-and-transposons-for-stable-gene-therapy-advances-current-challenges-and-perspectives
#10
José Eduardo Vargas, Leonardo Chicaybam, Renato Tetelbom Stein, Amilcar Tanuri, Andrés Delgado-Cañedo, Martin H Bonamino
Gene therapy protocols require robust and long-term gene expression. For two decades, retrovirus family vectors have offered several attractive properties as stable gene-delivery vehicles. These vectors represent a technology with widespread use in basic biology and translational studies that require persistent gene expression for treatment of several monogenic diseases. Immunogenicity and insertional mutagenesis represent the main obstacles to a wider clinical use of these vectors. Efficient and safe non-viral vectors are emerging as a promising alternative and facilitate clinical gene therapy studies...
October 12, 2016: Journal of Translational Medicine
https://www.readbyqxmd.com/read/27727248/homology-requirements-for-efficient-footprintless-gene-editing-at-the-cftr-locus-in-human-ipscs-with-helper-dependent-adenoviral-vectors
#11
Donna J Palmer, Nathan C Grove, Jordan Ing, Ana M Crane, Koen Venken, Brian R Davis, Philip Ng
Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb, helper-dependent adenoviral vectors with long homology arms are used for gene editing. However, this makes vector construction and recombinant analysis difficult. Conversely, insufficient homology may compromise targeting efficiency. Thus, we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation...
October 11, 2016: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/27701401/the-functionality-of-minimal-piggybac-transposons-in-mammalian-cells
#12
Boris Troyanovsky, Vira Bitko, Viktor Pastukh, Brian Fouty, Victor Solodushko
Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated...
October 4, 2016: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/27669309/transition-and-transversion-mutations-are-biased-towards-gc-in-transposons-of-chilo-suppressalis-lepidoptera-pyralidae
#13
Guang-Hua Luo, Xiao-Huan Li, Zhao-Jun Han, Zhi-Chun Zhang, Qiong Yang, Hui-Fang Guo, Ji-Chao Fang
Transposons are often regulated by their hosts, and as a result, there are transposons with several mutations within their host organisms. To gain insight into the patterns of the variations, nucleotide substitutions and indels of transposons were analysed in Chilo suppressalis Walker. The CsuPLE1.1 is a member of the piggyBac-like element (PLE) family, which belongs to the DNA transposons, and the Csu-Ty3 is a member of the Ty3/gypsy family, which belongs to the RNA transposons. Copies of CsuPLE1.1 and Csu-Ty3 were cloned separately from different C...
2016: Genes
https://www.readbyqxmd.com/read/27624004/efficient-production-of-fluorescent-transgenic-rats-using-the-piggybac-transposon
#14
Tianda Li, Ling Shuai, Junjie Mao, Xuepeng Wang, Mei Wang, Xinxin Zhang, Leyun Wang, Yanni Li, Wei Li, Qi Zhou
Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27621227/zfp462-deficiency-causes-anxiety-like-behaviors-with-excessive-self-grooming-in-mice
#15
B Wang, Y Zheng, H Shi, X Du, Y Zhang, B Wei, M Luo, H Wang, X Wu, X Hua, M Sun, X Xu
Zfp462 is a newly identified vertebrate-specific zinc finger protein that contains nearly 2500 amino acids and 23 putative C2H2-type zinc finger domains. So far, the functions of Zfp462 remain unclear. In our study, we showed that Zfp462 is expressed predominantly in the developing brain, especially in the cerebral cortex and hippocampus regions from embryonic day 7.5 to early postnatal stage. By using a piggyBac transposon-generated Zfp462 knockout (KO) mouse model, we found that Zfp462 KO mice exhibited prenatal lethality with normal neural tube patterning, whereas heterozygous (Het) Zfp462 KO (Zfp462(+/-) ) mice showed developmental delay with low body weight and brain weight...
September 13, 2016: Genes, Brain, and Behavior
https://www.readbyqxmd.com/read/27589724/the-piggybac-based-gene-delivery-system-can-confer-successful-production-of-cloned-porcine-blastocysts-with-multigene-constructs
#16
Masahiro Sato, Kosuke Maeda, Miyu Koriyama, Emi Inada, Issei Saitoh, Hiromi Miura, Masato Ohtsuka, Shingo Nakamura, Takayuki Sakurai, Satoshi Watanabe, Kazuchika Miyoshi
The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system...
2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27570481/analysis-of-pbase-binding-profile-indicates-an-insertion-target-selection-mechanism-dependent-on-ttaa-but-not-transcriptional-activity
#17
Dong Yang, Ruiqi Liao, Yun Zheng, Ling Sun, Tian Xu
Transposons and retroviruses are important pathogenic agents and tools for mutagenesis and transgenesis. Insertion target selection is a key feature for a given transposon or retrovirus. The piggyBac (PB) transposon is highly active in mice and human cells, which has a much better genome-wide distribution compared to the retrovirus and P-element. However, the underlying reason is not clear. Utilizing a tagged functional PB transposase (PBase), we were able to conduct genome-wide profiling for PBase binding sites in the mouse genome...
2016: International Journal of Biological Sciences
https://www.readbyqxmd.com/read/27565868/characterization-of-growth-and-reproduction-performance-transgene-integration-expression-and-transmission-patterns-in-transgenic-pigs-produced-by-piggybac-transposition-mediated-gene-transfer
#18
Fang Zeng, Zicong Li, Gengyuan Cai, Wenchao Gao, Gelong Jiang, Dewu Liu, Johann Urschitz, Stefan Moisyadi, Zhenfang Wu
Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs...
October 2016: Animal Biotechnology
https://www.readbyqxmd.com/read/27557691/seamless-genome-editing-in-rice-via-gene-targeting-and-precise-marker-elimination
#19
Ayako Nishizawa-Yokoi, Hiroaki Saika, Seiichi Toki
Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27530248/comparative-analysis-of-piggybac-crispr-cas9-and-talen-mediated-bac-transgenesis-in-the-zygote-for-the-generation-of-humanized-sirpa-rats
#20
Chris J Jung, Séverine Ménoret, Lucas Brusselle, Laurent Tesson, Claire Usal, Vanessa Chenouard, Séverine Remy, Laure-Hélène Ouisse, Nicolas Poirier, Bernard Vanhove, Pieter J de Jong, Ignacio Anegon
BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis...
2016: Scientific Reports
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