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https://www.readbyqxmd.com/read/29792157/long-term-health-and-germline-transmission-in-transgenic-cattle-following-transposon-mediated-gene-transfer
#1
Soo-Young Yum, Song-Jeon Lee, Sin-Gi Park, In-Gang Shin, Sang-Eun Hahn, Woo-Jae Choi, Hee-Soo Kim, Hyeong-Jong Kim, Seong-Hun Bae, Je-Hyeong Lee, Joo-Yeong Moon, Woo-Sung Lee, Ji-Hyun Lee, Choong-Il Lee, Seong-Jin Kim, Goo Jang
BACKGROUND: Transposon-mediated, non-viral gene delivery is a powerful tool for generating stable cell lines and transgenic animals. However, as multi-copy insertion is the preferred integration pattern, there is the potential for uncontrolled changes in endogenous gene expression and detrimental effects in cells or animals. Our group has previously reported on the generation of several transgenic cattle by using microinjection of the Sleeping Beauty (SB) and PiggyBac (PB) transposons and seeks to explore the long-term effects of this technology on cattle...
May 23, 2018: BMC Genomics
https://www.readbyqxmd.com/read/29784672/genetic-screening-and-multipotency-in-rhesus-monkey-haploid-neural-progenitor-cells
#2
Haisong Wang, Wenhao Zhang, Jian Yu, Congyu Wu, Qian Gao, Xu Li, Yanni Li, Jinxin Zhang, Yaru Tian, Tao Tan, Weizhi Ji, Luyuan Li, Yang Yu, Ling Shuai
Haploid embryonic stem cells (haESCs) have been extensively applied in forward and reverse genetic screening. However, the mammalian haploid somatic cell line is difficult to achieve because of spontaneous diploidization in differentiation. As a non-human primate species, monkeys are widely used in basic and pre-clinical research in which haploid cells are restricted to ESCs. Here, we report that rhesus monkey haESCs in an optimized culture medium showed naïve-state pluripotency and stable haploidy. This model facilitated the derivation of haploid neural progenitor cells (haNPCs), which maintained haploidy and differentiation potential into neurons and glia for a long period in vitro High-throughput trapping mutations can be efficiently introduced into haNPCs via piggyBac transposons...
May 21, 2018: Development
https://www.readbyqxmd.com/read/29782496/development-of-a-toolkit-for-piggybac-mediated-integrative-transfection-of-the-human-filarial-parasite-brugia-malayi
#3
Canhui Liu, Amruta S Mhashilkar, Johan Chabanon, Shulin Xu, Sara Lustigman, John H Adams, Thomas R Unnasch
BACKGROUND: The human filarial parasites cause diseases that are among the most important causes of morbidity in the developing world. The elimination programs targeting these infections rely on a limited number of drugs, making the identification of new chemotherapeutic agents a high priority. The study of these parasites has lagged due to the lack of reverse genetic methods. METHODOLOGY/PRINCIPAL FINDINGS: We report a novel co-culture method that results in developmentally competent infective larvae of one of the human filarial parasites (Brugia malayi) and describe a method to efficiently transfect the larval stages of this parasite...
May 21, 2018: PLoS Neglected Tropical Diseases
https://www.readbyqxmd.com/read/29750515/dimerization-through-the-ring-finger-domain-attenuates-excision-activity-of-the-piggybac-transposase
#4
Rahul Sharma, Shivlee Nirwal, Naveen Narayanan, Deepak T Nair
The movement of the piggyBac transposon is mediated through its cognate transposase. The piggyBac transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac transposase...
May 11, 2018: Biochemistry
https://www.readbyqxmd.com/read/29729503/direct-conversion-of-human-pluripotent-stem-cells-into-cranial-motor-neurons-using-a-piggybac-vector
#5
Riccardo De Santis, Maria Giovanna Garone, Francesca Pagani, Valeria de Turris, Silvia Di Angelantonio, Alessandro Rosa
Human pluripotent stem cells (PSCs) are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype...
April 27, 2018: Stem Cell Research
https://www.readbyqxmd.com/read/29725084/loss-of-tctn3-causes-neuronal-apoptosis-and-neural-tube-defects-in-mice
#6
Bin Wang, Yingying Zhang, Hongli Dong, Siyi Gong, Bin Wei, Man Luo, Hongyan Wang, Xiaohui Wu, Wei Liu, Xingshun Xu, Yufang Zheng, Miao Sun
Tctn3 belongs to the Tectonic (Tctn) family and is a single-pass membrane protein localized at the transition zone of primary cilia as an important component of ciliopathy-related protein complexes. Previous studies showed that mutations in Tctn1 and Tctn2, two members of the tectonic family, have been reported to disrupt neural tube development in humans and mice, but the functions of Tctn3 in brain development remain elusive. In this study, Tctn3 knockout (KO) mice were generated by utilizing the piggyBac (PB) transposon system...
May 3, 2018: Cell Death & Disease
https://www.readbyqxmd.com/read/29706119/nucleofection-with-plasmid-dna-for-crispr-cas9-mediated-inactivation-of-pd-1-in-cd133-specific-car-t-cells
#7
Bian Hu, Yan Zou, Linlin Zhang, Jiaxing Tang, Gabriele Niedermann, Elke Firat, Xingxu Huang, Xuekai Zhu
CRISPR/Cas9-mediated PD-1 disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of <i>in vitro</i> transcription of DNA or protein production...
April 28, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29687032/enhanced-expression-of-anti-cd19-chimeric-antigen-receptor-in-piggybac-transposon-engineered-t-cells
#8
Daisuke Morita, Nobuhiro Nishio, Shoji Saito, Miyuki Tanaka, Nozomu Kawashima, Yusuke Okuno, Satoshi Suzuki, Kazuyuki Matsuda, Yasuhiro Maeda, Matthew H Wilson, Gianpietro Dotti, Cliona M Rooney, Yoshiyuki Takahashi, Yozo Nakazawa
Adoptive T cell therapy using chimeric antigen receptor (CAR)-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs) as feeder cells and virus-specific T cell receptor (TCR) stimulation, we achieved 51...
March 16, 2018: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/29653176/hyperactive-piggybac-transposase-improves-transformation-efficiency-in-diverse-insect-species
#9
Kolja N Eckermann, Hassan M M Ahmed, Mohammad KaramiNejadRanjbar, Stefan Dippel, Christian E Ogaugwu, Peter Kitzmann, Musa D Isah, Ernst A Wimmer
Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites...
April 10, 2018: Insect Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/29645353/discovering-genes-responsible-for-silk-synthesis-in-bombyx-mori-by-piggybac-based-random-insertional-mutagenesis
#10
Xing-Bao Feng, Zi-Wen Zheng, Xian Zhang, Jun Gu, Qi-Li Feng, Li-Hua Huang
Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this mean, a "thin cocoon" mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) comparing with the wild type...
April 12, 2018: Insect Science
https://www.readbyqxmd.com/read/29627726/generation-of-wae001-a-15-a-human-embryonic-stem-cell-line-with-mir-122-doxycycline-inducible-expression
#11
Yanli Liu, Feima Wu, Dong Liu, Ruzhi Wei, Nasir Abbas, Jianhong Xia, Dongwei Li, Haiyun Wang, Yuanqi Zhuang, Dongsheng Guo, Yan Chen, Yuhang Wu, Xinrong Ke, Jiawang Tao, Fan Yang, Keyu Lai, Xiaodong Shu, Yin-Xiong Li
MiR-122 is the most abundant miRNA in the human liver accounting for 52% of the entire hepatic miRNome. Previous studies have demonstrated that miR-122 is a valuable therapeutic target for liver diseases, including viral hepatitis, fibrosis, steatosis, and hepatocarcinoma. Here, we constructed a miR-122 doxycycline-inducible expression human embryonic stem cell line WAe001-A-15 using the piggyBac transposon system. The cell line retained its pluripotency, in vitro differentiation potential, normal morphology, and karyotype...
March 23, 2018: Stem Cell Research
https://www.readbyqxmd.com/read/29625109/generation-of-splice-switching-oligonucleotides-targeting-the-cockayne-syndrome-group-b-gene-product-in-order-to-change-the-diseased-cell-state
#12
Yooksil Sin, Futaba Makimura, Masafumi Saijo, Satoshi Obika
Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UVS S), a disorder that causes mild symptoms. How the CSB gene determines a patient's fate is unknown, but one intriguing point is that in UVS S patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1...
April 3, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29587177/advances-in-functional-genetic-screening-with-transposons-and-crispr-cas9-to-illuminate-cancer-biology
#13
REVIEW
Kathryn A O'Donnell
Large-scale genome sequencing studies have identified a wealth of mutations in human tumors and have dramatically advanced the field of cancer genetics. However, the functional consequences of an altered gene in tumor progression cannot always be inferred from mutation status alone. This underscores the critical need for complementary methods to assign functional significance to mutated genes in cancer. Transposons are mobile genetic elements that serve as powerful tools for insertional mutagenesis. Over the last decade, investigators have employed mouse models with on-demand transposon-mediated mutagenesis to perform unbiased genetic screens to identify clinically relevant genes that participate in the pathogenesis of human cancer...
March 24, 2018: Current Opinion in Genetics & Development
https://www.readbyqxmd.com/read/29578046/multi-tissue-gal4-mediated-gene-expression-in-all-anopheles-gambiae-life-stages-using-an-endogenous-polyubiquitin-promoter
#14
Adriana Adolfi, Emilie Pondeville, Amy Lynd, Catherine Bourgouin, Gareth J Lycett
The ability to manipulate the Anopheles gambiae genome and alter gene expression effectively and reproducibly is a prerequisite for functional genetic analysis and for the development of novel control strategies in this important disease vector. However, in vivo transgenic analysis in mosquitoes is limited by the lack of promoters active ubiquitously. To address this, we used the GAL4/UAS system to investigate the promoter of the An. gambiae Polyubiquitin-c (PUBc) gene and demonstrated its ability to drive expression in mosquito cell culture before incorporation into An...
March 22, 2018: Insect Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/29572252/functional-and-clinical-relevance-of-novel-mutations-in-a-large-cohort-of-patients-with-cockayne-syndrome
#15
Nadege Calmels, Elena Botta, Nan Jia, Heather Fawcett, Tiziana Nardo, Yuka Nakazawa, Manuela Lanzafame, Shinichi Moriwaki, Katsuo Sugita, Masaya Kubota, Cathy Obringer, Marie-Aude Spitz, Miria Stefanini, Vincent Laugel, Donata Orioli, Tomoo Ogi, Alan Robert Lehmann
BACKGROUND: Cockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8 / CSA or ERCC6 / CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships...
March 23, 2018: Journal of Medical Genetics
https://www.readbyqxmd.com/read/29554589/conditional-gene-knockout-and-reconstitution-in-human-ipscs-with-an-inducible-cas9-system
#16
Mengyao Wu, Senquan Liu, Yongxing Gao, Hao Bai, Vasiliki Machairaki, Gang Li, Tong Chen, Linzhao Cheng
Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome...
March 10, 2018: Stem Cell Research
https://www.readbyqxmd.com/read/29503200/autologous-cell-therapy-approach-for-duchenne-muscular-dystrophy-using-piggybac-transposons-and-mesoangioblasts
#17
Pavithra S Iyer, Lionel O Mavoungou, Flavio Ronzoni, Joanna Zemla, Emanuel Schmid-Siegert, Stefania Antonini, Laurence A Neff, Olivier M Dorchies, Marisa Jaconi, Malgorzata Lekka, Graziella Messina, Nicolas Mermod
Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression...
April 4, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/29493878/developing-a-piggybac-transposon-system-and-compatible-selection-markers-for-insertional-mutagenesis-and-genome-engineering-in-yarrowia-lipolytica
#18
James M Wagner, Eden V Williams, Hal S Alper
Yarrowia lipolytica is a non-conventional yeast of interest to the biotechnology industry. However, the physiology, metabolism, and genetic regulation of Y. lipolytica diverge significantly from more well-studied and characterized yeasts such as Saccharomyces cerevisiae. To develop additional genetic tools for this industrially relevant host, the piggyBac transposon system to enable efficient generation of genome-wide insertional mutagenesis libraries and introduction of scarless, footprint-free genomic modifications in Y...
May 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29484202/fusion-of-piggybac-like-transposons-and-herpesviruses-occurs-frequently-in-teleosts
#19
Yusuke Inoue, Masahiko Kumagai, Xianbo Zhang, Tomonori Saga, Deshou Wang, Akihiko Koga, Hiroyuki Takeda
Background: Endogenous viral elements play important roles in eukaryotic evolution by giving rise to genetic novelties. Herpesviruses are a large family of DNA viruses, most of which do not have the ability to endogenize into host genomes. Recently, we identified a novel type of endogenous herpesvirus, which we named " Teratorn ", from the medaka ( Oryzias latipes ) genome, in which the herpesvirus is fused with a piggyBac -like DNA transposon, forming a novel mobile element...
2018: Zoological Letters
https://www.readbyqxmd.com/read/29475789/a-new-approach-to-car-t-cell-gene-engineering-and-cultivation-using-piggybac-transposon-in-the-presence-of-il-4-il-7-and-il-21
#20
Pavlína Ptáčková, Jan Musil, Martin Štach, Petr Lesný, Šárka Němečková, Vlastimil Král, Milan Fábry, Pavel Otáhal
BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21...
April 2018: Cytotherapy
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