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https://www.readbyqxmd.com/read/28934495/a-germline-limited-piggybac-transposase-gene-is-required-for-precise-excision-in-tetrahymena-genome-rearrangement
#1
Lifang Feng, Guangying Wang, Eileen P Hamilton, Jie Xiong, Guanxiong Yan, Kai Chen, Xiao Chen, Wen Dui, Amber Plemens, Lara Khadr, Arjune Dhanekula, Mina Juma, Hung Quang Dang, Geoffrey M Kapler, Eduardo Orias, Wei Miao, Yifan Liu
Developmentally programmed genome rearrangement accompanies differentiation of the silent germline micronucleus into the transcriptionally active somatic macronucleus in the ciliated protozoan Tetrahymena thermophila. Internal eliminated sequences (IES) are excised, followed by rejoining of MAC-destined sequences, while fragmentation occurs at conserved chromosome breakage sequences, generating macronuclear chromosomes. Some macronuclear chromosomes, referred to as non-maintained chromosomes (NMC), are lost soon after differentiation...
September 19, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28920716/transposons-moving-forward-from-preclinical-studies-to-clinical-trials
#2
Jaitip Tipanee, Thierry VandenDriessche, Marinee K Chuah
Transposons have emerged as promising vectors for gene therapy that can potentially overcome some of the limitations of commonly used viral vectors. Transposons stably integrate into the target cell genome, enabling persistent expression of therapeutic genes. Transposons have evolved from being used as basic tools in biomedical research to bona fide therapeutics. Currently, the most promising transposons for gene therapy applications are derived from Sleeping Beauty (SB) or piggyBac (PB). Stable transposition requires co-delivery of the transposon DNA with the corresponding transposase gene, mRNA, or protein...
August 22, 2017: Human Gene Therapy
https://www.readbyqxmd.com/read/28918057/one-step-piggybac-transposon-based-crispr-cas9-activation-of-multiple-genes
#3
Shenglan Li, Anqi Zhang, Haipeng Xue, Dali Li, Ying Liu
Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs...
September 15, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28916771/complete-fusion-of-a-transposon-and-herpesvirus-created-the-teratorn-mobile-element-in-medaka-fish
#4
Yusuke Inoue, Tomonori Saga, Takumi Aikawa, Masahiko Kumagai, Atsuko Shimada, Yasushi Kawaguchi, Kiyoshi Naruse, Shinichi Morishita, Akihiko Koga, Hiroyuki Takeda
Mobile genetic elements (e.g., transposable elements and viruses) display significant diversity with various life cycles, but how novel elements emerge remains obscure. Here, we report a giant (180-kb long) transposon, Teratorn, originally identified in the genome of medaka, Oryzias latipes. Teratorn belongs to the piggyBac superfamily and retains the transposition activity. Remarkably, Teratorn is largely derived from a herpesvirus of the Alloherpesviridae family that could infect fish and amphibians. Genomic survey of Teratorn-like elements reveals that some of them exist as a fused form between piggyBac transposon and herpesvirus genome in teleosts, implying the generality of transposon-herpesvirus fusion...
September 15, 2017: Nature Communications
https://www.readbyqxmd.com/read/28854370/generation-of-mouse-haploid-somatic-cells-by-small-molecules-for-genome-wide-genetic-screening
#5
Zheng-Quan He, Bao-Long Xia, Yu-Kai Wang, Jing Li, Gui-Hai Feng, Lin-Lin Zhang, Yu-Huan Li, Hai-Feng Wan, Tian-Da Li, Kai Xu, Xue-Wei Yuan, Yu-Fei Li, Xin-Xin Zhang, Ying Zhang, Liu Wang, Wei Li, Qi Zhou
The recent success of derivation of mammalian haploid embryonic stem cells (haESCs) has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons...
August 29, 2017: Cell Reports
https://www.readbyqxmd.com/read/28798873/a-novel-viral-lineage-distantly-related-to-herpesviruses-discovered-within-fish-genome-sequence-data
#6
Amr Aswad, Aris Katzourakis
Pathogenic viruses represent a small fraction of viral diversity, and emerging diseases are frequently the result of cross-species transmissions. Therefore, we need to develop high-throughput techniques to investigate a broader range of viral biodiversity across a greater number of species. This is especially important in the context of new practices in agriculture that have arisen to tackle the challenges of global food security, including the rising number of marine and freshwater species that are used in aquaculture...
July 2017: Virus Evolution
https://www.readbyqxmd.com/read/28753244/functional-characterization-of-the-transcriptional-regulatory-elements-of-three-highly-expressed-constitutive-genes-in-the-jewel-wasp-nasonia-vitripennis
#7
P D Shirk, R B Furlong, A Dolan, J H Werren
The jewel wasp, Nasonia vitripennis Ashmead (Hymenoptera: Pteromalidae), is an easily reared parasitoid that is providing an ever increasingly malleable model for examining the biology and genetics of Hymenoptera. Utilizing genomic and transcriptome resources, 5' upstream transcriptional regulatory sequences (TREs) from three highly expressed genes were identified and cloned. Criteria for TRE selection included the presence of an adjacent gene 5' of the translation initiation site. One gene was methylated whereas the other two were nonmethylated...
July 28, 2017: Insect Molecular Biology
https://www.readbyqxmd.com/read/28720304/characterization-of-the-single-cell-derived-bovine-induced-pluripotent-stem-cells
#8
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
#9
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...
August 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
#10
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...
August 21, 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
#11
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
#12
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...
July 11, 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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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