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https://www.readbyqxmd.com/read/28720304/characterization-of-the-single-cell-derived-bovine-induced-pluripotent-stem-cells
#1
Lixia Zhao, Zixin Wang, Jindun Zhang, Jian Yang, Xuefei Gao, Baojiang Wu, Gaoping Zhao, Siqin Bao, Shuxiang Hu, Pentao Liu, Xihe Li
Single-cell derived bovine induced pluripotent stem cells (iPSCs) were generated by the introduction of piggyBac transposons with CAG promoting transcription factors (Oct3/4, Sox2, Klf4 and cMyc). In the study, the bovine iPSCs colony from single cell could passage more than 50 passages after enzymatic dissociation into single cells. These bovine iPSCs cells kept the normal karyotype and displayed dome shaped clones similar to mouse embryonic stem cells. They showed pluripotency in many ways, including their expression of pluripotency markers, such as OCT3/4, NANOG, SOX2, SSEA1, SSEA4, and AP in immunofluorescence assay, Oct4, Nanog, Sox2, Klf4 and cMyc in RT-PCR...
May 22, 2017: Tissue & Cell
https://www.readbyqxmd.com/read/28676355/nanos-driven-expression-of-piggybac-transposase-induces-mobilization-of-a-synthetic-autonomous-transposon-in-the-malaria-vector-mosquito-anopheles-stephensi
#2
Vanessa M Macias, Alyssa J Jimenez, Bianca Burini-Kojin, David Pledger, Nijole Jasinskiene, Celine Hien Phong, Karen Chu, Aniko Fazekas, Kelcie Martin, Osvaldo Marinotti, Anthony A James
Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos...
July 1, 2017: Insect Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/28666380/comparative-analysis-of-chimeric-zfp-tale-and-cas9-piggybac-transposases-for-integration-into-a-single-locus-in-human-cells
#3
Wentian Luo, Daniel L Galvan, Lauren E Woodard, Dan Dorset, Shawn Levy, Matthew H Wilson
Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X...
June 29, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28662065/monoclonal-antibodies-expression-improvement-in-cho-cells-by-piggybac-transposition-regarding-vectors-ratios-and-design
#4
Samira Ahmadi, Fatemeh Davami, Noushin Davoudi, Fatemeh Nematpour, Maryam Ahmadi, Saeedeh Ebadat, Kayhan Azadmanesh, Farzaneh Barkhordari, Fereidoun Mahboudi
Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb...
2017: PloS One
https://www.readbyqxmd.com/read/28645898/dna-pk-facilitates-piggybac-transposition-by-promoting-paired-end-complex-formation
#5
Yan Jin, Yaohui Chen, Shimin Zhao, Kun-Liang Guan, Yuan Zhuang, Wenhao Zhou, Xiaohui Wu, Tian Xu
The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively...
June 23, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28643257/genome-editing-mediated-by-primordial-germ-cell-in-chicken
#6
Jae Yong Han, Hong Jo Lee
Rapid development of genome editing technology has facilitated the studies on exploring specific gene functions and establishment of model animals. In livestock, the technology has contributed to create high value in industry fields, e.g., enhancing productivity or acquiring the resistance against disease. Meanwhile, genome editing in avian species has been emphasized because of their applicable possibilities in terms of highly productive chickens, disease-controlled avian lines, and development of novel biological models...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28642584/molecular-switching-system-using-glycosylphosphatidylinositol-to-select-cells-highly-expressing-recombinant-proteins
#7
Emmanuel Matabaro, Zeng'an He, Yi-Shi Liu, Hui-Jie Zhang, Xiao-Dong Gao, Morihisa Fujita
Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport...
June 22, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28610919/neural-differentiation-of-human-embryonic-stem-cells-induced-by-the-transgene-mediated-overexpression-of-single-transcription-factors
#8
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...
August 19, 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
#9
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
#10
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
#11
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
#12
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
#13
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
#14
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...
July 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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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