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https://www.readbyqxmd.com/read/28610919/neural-differentiation-of-human-embryonic-stem-cells-induced-by-the-transgene-mediated-overexpression-of-single-transcription-factors
#1
Misako Matsushita, Yuhki Nakatake, Itaru Arai, Keiji Ibata, Kazuhisa Kohda, Sravan K Goparaju, Miyako Murakami, Miki Sakota, Nana Chikazawa-Nohtomi, Shigeru B H Ko, Takanori Kanai, Michisuke Yuzaki, Minoru S H Ko
Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons...
June 10, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28583832/yorkie-ca-overexpression-in-the-posterior-silk-gland-improves-silk-yield-in-bombyx-mori
#2
Panli Zhang, Shumin Liu, Hong-Sheng Song, Guozheng Zhang, Qiangqiang Jia, Sheng Li
The traditional hybrid breeding techniques can no longer meet the increasing demands for silk production by the silkworm, Bombyx mori, and further improvement of the silk yield will depend on modern molecular breeding techniques. Here, we report improved silk yield in transgenic silkworms overexpressing the oncogene Yorkie(CA) specifically in the posterior silk gland (PSG). The Yorkie(CA) cDNA was ligated downstream of the hr3 enhancer and the fibroin L-chain (Fil) promoter, then inserted into a piggyBac vector for transgene...
June 2, 2017: Journal of Insect Physiology
https://www.readbyqxmd.com/read/28566761/proof-of-concept-gene-editing-for-the-murine-model-of-inducible-arginase-1-deficiency
#3
Yuan Yan Sin, Phillipe R Price, Laurel L Ballantyne, Colin D Funk
Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro...
May 31, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28547769/evaluation-of-piggybac-mediated-cho-pools-to-enable-material-generation-to-support-glp-toxicology-studies
#4
Yashas Rajendra, Sowmya Balasubramanian, Neil A McCracken, Dawn L Norris, Zhirui Lian, Matthew G Schmitt, Christopher C Frye, Gavin C Barnard
Generating purified protein for GLP toxicology studies (GLP-Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100g), typically produced in a single 500L - 2,500L bioreactor, using the final CHO clonally-derived cell line (CDCL). One approach currently used to save time is to manufacture GLP-Tox material using pools of high-producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers >7 g/L using piggyBac-mediated gene integration (PB CHO pools)...
May 25, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28533524/enhancing-the-genome-editing-toolbox-genome-wide-crispr-arrayed-libraries
#5
Emmanouil Metzakopian, Alex Strong, Vivek Iyer, Alex Hodgkins, Konstantinos Tzelepis, Liliana Antunes, Mathias J Friedrich, Qiaohua Kang, Teresa Davidson, Jacob Lamberth, Christina Hoffmann, Gregory D Davis, George S Vassiliou, William C Skarnes, Allan Bradley
CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers...
May 22, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28510333/tetracycline-inducible-and-reversible-stable-gene-expression-in-human-ipsc-derived-neural-progenitors-and-in-the-postnatal-mouse-brain
#6
Aslam Abbasi Akhtar, Joshua J Breunig
The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28504702/pgbd5-promotes-site-specific-oncogenic-mutations-in-human-tumors
#7
Anton G Henssen, Richard Koche, Jiali Zhuang, Eileen Jiang, Casie Reed, Amy Eisenberg, Eric Still, Ian C MacArthur, Elias Rodríguez-Fos, Santiago Gonzalez, Montserrat Puiggròs, Andrew N Blackford, Christopher E Mason, Elisa de Stanchina, Mithat Gönen, Anne-Katrin Emde, Minita Shah, Kanika Arora, Catherine Reeves, Nicholas D Socci, Elizabeth Perlman, Cristina R Antonescu, Charles W M Roberts, Hanno Steen, Elizabeth Mullen, Stephen P Jackson, David Torrents, Zhiping Weng, Scott A Armstrong, Alex Kentsis
Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes...
May 15, 2017: Nature Genetics
https://www.readbyqxmd.com/read/28500952/isolation-and-characterization-of-lymphoid-enhancer-factor-1-positive-deciduous-dental-pulp-stem-like-cells-after-transfection-with-a-piggybac-vector-containing-lef1-promoter-driven-selection-markers
#8
Tomoya Murakami, Issei Saitoh, Masahiro Sato, Emi Inada, Miki Soda, Masataka Oda, Hisanori Domon, Yoko Iwase, Tadashi Sawami, Kazunari Matsueda, Yutaka Terao, Hayato Ohshima, Hirofumi Noguchi, Haruaki Hayasaki
OBJECTIVE: Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter...
May 7, 2017: Archives of Oral Biology
https://www.readbyqxmd.com/read/28491150/dynamic-silencing-of-somatic-l1-retrotransposon-insertions-reflects-the-developmental-and-cellular-contexts-of-their-genomic-integration
#9
Manoj Kannan, Jingfeng Li, Sarah E Fritz, Kathryn E Husarek, Jonathan C Sanford, Teresa L Sullivan, Pawan Kumar Tiwary, Wenfeng An, Jef D Boeke, David E Symer
BACKGROUND: The ongoing mobilization of mammalian transposable elements (TEs) contributes to natural genetic variation. To survey the epigenetic control and expression of reporter genes inserted by L1 retrotransposition in diverse cellular and genomic contexts, we engineered highly sensitive, real-time L1 retrotransposon reporter constructs. RESULTS: Here we describe different patterns of expression and epigenetic controls of newly inserted sequences retrotransposed by L1 in various somatic cells and tissues including cultured human cancer cells, mouse embryonic stem cells, and tissues of pseudofounder transgenic mice and their progeny...
2017: Mobile DNA
https://www.readbyqxmd.com/read/28484230/an-all-in-one-tet-on-3g-inducible-piggybac-system-for-human-pluripotent-stem-cells-and-derivatives
#10
Lauren N Randolph, Xiaoping Bao, Chikai Zhou, Xiaojun Lian
Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner...
May 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28463838/engineering-the-rapid-adenovirus-production-and-amplification-rapa-cell-line-to-expedite-the-generation-of-recombinant-adenoviruses
#11
Qiang Wei, Jiaming Fan, Junyi Liao, Yulong Zou, Dongzhe Song, Jianxiang Liu, Jing Cui, Feng Liu, Chao Ma, Xue Hu, Li Li, Yichun Yu, Xiangyang Qu, Liqun Chen, Xinyi Yu, Zhicai Zhang, Chen Zhao, Zongyue Zeng, Ruyi Zhang, Shujuan Yan, Xingye Wu, Yi Shu, Russell R Reid, Michael J Lee, Jennifer Moritis Wolf, Tong-Chuan He
BACKGROUND/AIMS: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. METHODS: Stable transgene expression in 293T cells was accomplished by using piggyBac system...
2017: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/28428837/large-is-required-for-normal-astrocyte-migration-and-retinal-vasculature-development
#12
Min Zhou, Herui Wang, Hui Ren, Rui Jiang, Chi Zhang, Xiaohui Wu, Gezhi Xu
BACKGROUND: Persistent fetal vasculature (PFV) is a congenital developmental anomaly of the eye that accounts for about 5% of childhood blindness. The molecular mechanism of PFV remains unclear. As a glycosyltransferase of α-dystroglycan, LARGE mutations have been found in congenital muscular dystrophy patients with brain abnormalities. Spontaneous Large mutant mice displayed similar symptoms of human muscle-eye-brain disorders. However, the detailed roles of Large in ocular vasculature development still need to be uncovered...
2017: Cell & Bioscience
https://www.readbyqxmd.com/read/28397990/germline-transformation-of-the-western-corn-rootworm-diabrotica-virgifera-virgifera
#13
F Chu, W Klobasa, P Wu, S Pinzi, N Grubbs, S Gorski, Y Cardoza, M D Lorenzen
The western corn rootworm (WCR), a major pest of maize, is notorious for rapidly adapting biochemically, behaviourally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Since transformation-based applications such as transposon tagging and enhancer trapping have facilitated genetic dissection of model species such as Drosophila melanogaster, we developed a germline-transformation system for WCR in an effort to gain a greater understanding of the basic biology of this economically important insect...
April 11, 2017: Insect Molecular Biology
https://www.readbyqxmd.com/read/28381173/mesothelin-targeting-chimeric-antigen-receptor-modified-t-cells-by-piggybac-transposon-system-suppress-the-growth-of-bile-duct-carcinoma
#14
Jie-Ying Xu, Zhen-Long Ye, Du-Qing Jiang, Jiang-Chuan He, Yong-Mei Ding, Lin-Fang Li, Sai-Qun Lv, Ying Wang, Hua-Jun Jin, Qi-Jun Qian
Chimeric antigen receptor modified T cell-based immunotherapy is revolutionizing the field of cancer treatment. However, its potential in treating bile duct carcinoma has not been fully explored. Herein, we developed the second-generation mesothelin-targeting chimeric antigen receptor-modified T cells with the 4-1BB co-stimulatory module by the piggyBac transposon system. Mesothelin-targeting chimeric antigen receptor was expressed by 66.0% of mesothelin-targeting chimeric antigen receptor-modified T cells post electrophoretic transfection and stimulation with K562-meso cells; the expressions of activation markers were tested by flow cytometry assay and showed greater activation of mesothelin-targeting chimeric antigen receptor-modified T cells than control T cells (CD107α: 71...
April 2017: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
https://www.readbyqxmd.com/read/28325684/monitoring-and-visualizing-microrna-dynamics-during-live-cell-differentiation-using-microrna-responsive-non-viral-reporter-vectors
#15
Hideyuki Nakanishi, Kenji Miki, Kaoru R Komatsu, Masayuki Umeda, Megumi Mochizuki, Azusa Inagaki, Yoshinori Yoshida, Hirohide Saito
MicroRNA (miRNA) activity differs with cell type, suggesting it can be used as a cell marker. In this study, we developed novel miRNA-responsive non-viral reporter vectors to continuously monitor and visualize miRNA dynamics during differentiation and to efficiently purify target living cells. Each vector codes miRNA-responsive and reference reporter genes in a single mRNA. These two genes are independent modules but transcribed by a single promoter, which enables us to distinguish miRNA-mediated post-transcriptional repression from transcriptional repression...
March 2, 2017: Biomaterials
https://www.readbyqxmd.com/read/28317878/kidney-specific-transposon-mediated-gene-transfer-in-vivo
#16
Lauren E Woodard, Jizhong Cheng, Richard C Welch, Felisha M Williams, Wentian Luo, Leslie S Gewin, Matthew H Wilson
Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues...
March 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28295042/generation-of-non-viral-transgene-free-hepatocyte-like-cells-with-piggybac-transposon
#17
Hokahiro Katayama, Kentaro Yasuchika, Yuya Miyauchi, Hidenobu Kojima, Ryoya Yamaoka, Takayuki Kawai, Elena Yukie Yoshitoshi, Satoshi Ogiso, Sadahiko Kita, Katsutaro Yasuda, Naoya Sasaki, Ken Fukumitsu, Junji Komori, Takamichi Ishii, Shinji Uemoto
Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method...
March 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28280212/genetic-and-transgenic-reagents-for-drosophila-simulans-d-mauritiana-d-yakuba-d-santomea-and-d-virilis
#18
David L Stern, Justin Crocker, Yun Ding, Nicolas Frankel, Gretchen Kappes, Elizabeth Kim, Ryan Kuzmickas, Andrew Lemire, Joshua D Mast, Serge Picard
Species of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. However, studies in these species have been limited by a paucity of genetic and transgenic reagents. Here, we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein (EYFP) gene expressed in the eyes and a ϕC31 attP site-specific integration site...
April 3, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28265066/resistance-mechanisms-to-tp53-mdm2-inhibition-identified-by-in-vivo-piggybac-transposon-mutagenesis-screen-in-an-arf-mouse-model
#19
Emilie A Chapeau, Agnieszka Gembarska, Eric Y Durand, Emeline Mandon, Claire Estadieu, Vincent Romanet, Marion Wiesmann, Ralph Tiedt, Joseph Lehar, Antoine de Weck, Roland Rad, Louise Barys, Sebastien Jeay, Stephane Ferretti, Audrey Kauffmann, Esther Sutter, Armelle Grevot, Pierre Moulin, Masato Murakami, William R Sellers, Francesco Hofmann, Michael Rugaard Jensen
Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201...
March 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28257857/generation-of-a-transgenic-cashmere-goat-using-the-piggybac-transposition-system
#20
Ding-Ping Bai, Ming-Ming Yang, Lei Qu, Yu-Lin Chen
The development of transgenic technologies in the Cashmere goat (Capra hircus) has the potential to improve the quality of the meat and wool. The piggyBac (PB) transposon system is highly efficient and can be used to transpose specific target genes into the genome. Here, we developed a PB transposon system to produce transgenic Cashmere goat fetal fibroblasts (GFFs) with the enhanced green fluorescent protein (EGFP). We then used the genetically modified GFFs as nuclear donors to generate transgenic embryos by somatic cell nuclear transfer (SCNT)...
April 15, 2017: Theriogenology
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