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https://www.readbyqxmd.com/read/29777593/degradation-of-recombinant-proteins-by-cho-host-cell-proteases-is-prevented-by-matriptase-1-knock-out
#1
Holger Laux, Sandrine Romand, Sandro Nuciforo, Christopher J Farady, Joel Tapparel, Stine Buechmann-Moeller, Benjamin Sommer, Edward J Oakeley, Ursula Bodendorf
An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells...
May 19, 2018: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29769555/cattle-with-a-precise-zygote-mediated-deletion-safely-eliminate-the-major-milk-allergen-beta-lactoglobulin
#2
Jingwei Wei, Stefan Wagner, Paul Maclean, Brigid Brophy, Sally Cole, Grant Smolenski, Dan F Carlson, Scott C Fahrenkrug, David N Wells, Götz Laible
We applied precise  zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows' milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves...
May 16, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29766738/the-role-of-gene-editing-in-neurodegenerative-diseases
#3
Hueng-Chuen Fan, Ching-Shiang Chi, Yih-Jing Lee, Jeng-Dau Tsai, Shinn-Zong Lin, Horng-Jyh Harn
Neurodegenerative diseases (NDs), at least including Alzheimer's, Huntington's, and Parkinson's diseases, have become the most dreaded maladies because there are no precise diagnostic tools or definite treatments for these debilitating diseases. The increased prevalence and a substantial impact on the social-economic and medical care of NDs propel governments to develop policies to counteract the impact. Although the etiologies of NDs are still unknown, growing evidence suggests that genetic, cellular, and circuit alternations may cause the generation of abnormal misfolded proteins, which uncontrolledly accumulate to damage and eventually overwhelm the protein-disposal mechanisms of these neurons, leading to a common pathological feature of NDs...
January 1, 2018: Cell Transplantation
https://www.readbyqxmd.com/read/29754773/genome-editing-in-plants-advancing-crop-transformation-and-overview-of-tools
#4
Tariq Shah, Tayyaba Andleeb, Sadia Lateef, Mehmood Ali Noor
Genome manipulation technology is one of emerging field which brings real revolution in genetic engineering and biotechnology. Targeted editing of genomes pave path to address a wide range of goals not only to improve quality and productivity of crops but also permit to investigate the fundamental roots of biological systems. These goals includes creation of plants with valued compositional properties and with characters that confer resistance to numerous biotic and abiotic stresses. Numerous novel genome editing systems have been introduced during the past few years; these comprise zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9)...
May 7, 2018: Plant Physiology and Biochemistry: PPB
https://www.readbyqxmd.com/read/29754248/star-a-simple-tal-effector-assembly-reaction-using-isothermal-assembly
#5
Sabine Gogolok, Ute Köber, Steven M Pollard
Transcription activator-like effectors (TALEs) contain programmable DNA-binding domains that can be fused to various effectors to manipulate genetic sequences or transcriptional state. However, the construction of plasmids encoding the modular DNA-binding domain remains challenging due to their repetitive nature. Here, we describe methods for a simple TALE assembly reaction (STAR) that uses a 68-part plasmid library to create TALEs binding to 17 bp target sequences. Manual production of many tens of TALEs can be achieved using a simple 8 h protocol, with full length sequence-verified plasmids available within a few days...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29730572/establishment-of-a-human-embryonic-stem-cell-line-with-homozygous-tp53-r248w-mutant-by-talen-mediated-gene-editing
#6
An Xu, Ruoji Zhou, Jian Tu, Zijun Huo, Dandan Zhu, Donghui Wang, Julian A Gingold, Helen Mata, Pulivarthi H Rao, Mo Liu, Alaa M T Mohamed, Celine Shuet Lin Kong, Brittany E Jewell, Weiya Xia, Ruiying Zhao, Mien-Chie Hung, Dung-Fang Lee
Genetic mutations in TP53 contribute to multiple human cancers. Here we report the generation of a H1-p53(R248W/R248W) human embryonic stem cell line harboring a homozygous TP53 R248W mutation created by TALEN-mediated precise gene editing. The H1-p53(R248W/R248W) cell line maintains a normal karyotype, robust pluripotency gene expression, and the potential to differentiate to the three germ layers.
April 27, 2018: Stem Cell Research
https://www.readbyqxmd.com/read/29723918/genotyping-genome-edited-mutations-in-plants-using-crispr-ribonucleoprotein-complexes
#7
Zhen Liang, Kunling Chen, Yan Yan, Yi Zhang, Caixia Gao
Despite the great achievements in genome editing, accurately detecting mutations induced by sequence-specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome-edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site...
May 3, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29721989/the-retinol-binding-protein-receptor-2-rbpr2-is-required-for-photoreceptor-survival-and-visual-function-in-the-zebrafish
#8
Glenn P Lobo, Gayle Pauer, Joshua H Lipschutz, Stephanie A Hagstrom
Vitamin A/retinol (ROL) and its metabolites (retinoids) play critical roles in eye development and photoreception. Short-term dietary vitamin A deficiency (VAD) manifests clinically as night blindness, while prolonged VAD is known to cause retinal pigment epithelium (RPE) and photoreceptor degeneration. Therefore, sustained uptake of dietary vitamin A, for ocular retinoid production, is essential for photoreceptor health and visual function. The mechanisms influencing the uptake, storage, and supply of dietary vitamin A, for ocular retinoid production, however, are not fully understood...
2018: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/29720730/a-proteomic-analysis-of-an-in-vitro-knock-out-of-mir-200c
#9
Bojan Ljepoja, Jonathan García-Roman, Ann-Katrin Sommer, Thomas Fröhlich, Georg J Arnold, Ernst Wagner, Andreas Roidl
Loss of miR-200c is correlated to advanced cancer-subtypes due to increased EMT and decreased treatment efficacy by chemotherapeutics. As miRNAs regulate a multitude of targets, the analysis of differentially expressed proteins upon a genomic knock-out (KO) is of interest. In this study, we generated a TALENs KO of miR-200c in MCF7 breast cancer cells, excluded its compensation by family-members and evaluated the impact on the proteome by analyzing three individual KO-clones. We identified 26 key proteins and a variety of enrichments in metabolic and cytoskeletal pathways...
May 2, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29718583/targeted-gene-disruption-by-use-of-crispr-cas9-ribonucleoprotein-complexes-in-the-water-flea-daphnia-pulex
#10
Chizue Hiruta, Keiichi Kakui, Knut E Tollefsen, Taisen Iguchi
The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D...
May 2, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
https://www.readbyqxmd.com/read/29701183/-in-vitro-study-of-joint-intervention-of-e-cad-and-bmi-1-mediated-by-transcription-activator-like-effector-nuclease-in-nasopharyngeal-carcinoma
#11
Tingting Luo, Aifen Yan, Lian Liu, Hong Jiang, Cuilan Feng, Guannan Liu, Fang Liu, Dongsheng Tang, Tianhong Zhou
To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells.
 Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously...
March 28, 2018: Zhong Nan da Xue Xue Bao. Yi Xue Ban, Journal of Central South University. Medical Sciences
https://www.readbyqxmd.com/read/29700675/talen-mediated-targeted-mutagenesis-of-fatty-acid-desaturase-2-fad2-in-peanut-arachis-hypogaea-l-promotes-the-accumulation-of-oleic-acid
#12
Shijie Wen, Hao Liu, Xingyu Li, Xiaoping Chen, Yanbin Hong, Haifen Li, Qing Lu, Xuanqiang Liang
A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut...
April 26, 2018: Plant Molecular Biology
https://www.readbyqxmd.com/read/29696633/generation-of-reporter-hescs-by-targeting-egfp-at-the-cd144-locus-to-facilitate-the-endothelial-differentiation
#13
Zhiqing Hu, Yong Wu, Miaojin Zhou, Xiaolin Wang, Jialun Pang, Zhuo Li, Mai Feng, Yanchi Wang, Qian Hu, Junya Zhao, Xionghao Liu, Lingqian Wu, Desheng Liang
Reporter embryonic stem cell (ESC) lines with tissue-specific reporter genes may contribute to optimizing the differentiation conditions in vitro as well as trafficking transplanted cells in vivo. To optimize and monitor endothelial cell (EC) differentiation specifically, here we targeted the enhanced green fluorescent protein (EGFP) reporter gene at the junction of 5'UTR and exon2 of the endothelial specific marker gene CD144 using TALENs in human ESCs (H9) to generate a EGFP-CD144-reporter ESC line. The reporter cells expressed EGFP and CD144 increasingly and specifically without unexpected effects during the EC differentiation...
April 25, 2018: Development, Growth & Differentiation
https://www.readbyqxmd.com/read/29685818/strategies-for-in-vivo-genome-editing-in-nondividing-cells
#14
REVIEW
Fatemeharefeh Nami, Mohsen Basiri, Leila Satarian, Cameron Curtiss, Hossein Baharvand, Catherine Verfaillie
Programmable nucleases, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have enhanced our ability to edit genomes by the sequence-specific generation of double-strand breaks (DSBs) with subsequent homology-directed repair (HDR) of the DSB. However, the efficiency of the HDR pathway is limited in nondividing cells, which encompass most of the cells in the body. Therefore, the HDR-mediated genome-editing approach has limited in vivo applicability...
April 21, 2018: Trends in Biotechnology
https://www.readbyqxmd.com/read/29681919/efficient-gene-transfer-and-gene-editing-in-sterlet-acipenser-ruthenus
#15
Ji Chen, Wei Wang, Zhaohui Tian, Ying Dong, Tian Dong, Hua Zhu, Zuoyan Zhu, Hongxia Hu, Wei Hu
The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet ( Acipenser ruthenus ), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved...
2018: Frontiers in Genetics
https://www.readbyqxmd.com/read/29678790/development-of-enzymatically-active-bacterial-cellulose-membranes-through-stable-immobilization-of-an-engineered-%C3%AE-galactosidase
#16
Berta N Estevinho, Nuria Samaniego, David Talens-Perales, Maria José Fabra, Amparo López-Rubio, Julio Polaina, Julia Marín-Navarro
Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme...
April 17, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29678510/corrigendum-to-dual-chemotactic-factors-secreting-human-amniotic-mesenchymal-stem-cells-via-talen-mediated-gene-editing-enhanced-angiogenesis-int-j-cardiol-260-2018-156-162
#17
In Sil Jeong, Youngjin Park, Hyun Aae Ryu, Hyun Sook An, Ju Hye Han, Sung-Whan Kim
No abstract text is available yet for this article.
April 18, 2018: International Journal of Cardiology
https://www.readbyqxmd.com/read/29672693/metabolic-and-genomic-characterization-of-stress-tolerant-industrial-saccharomyces-cerevisiae-strains-from-talens-assisted-multiplex-editing
#18
Yuman Gan, Yuping Lin, Yufeng Guo, Xianni Qi, Qinhong Wang
TALENs-assisted multiplex editing (TAME) toolbox was previously established and used to successfully enhance ethanol-stress tolerance of S. cerevisiae laboratory strain. Here, the TAME toolbox was harnessed to improve and elucidate stress tolerances of S. cerevisiae industrial strain. One osmotolerant strain and one thermotolerant strain were selected from the mutant library generated by TAME at corresponding stress conditions, and exhibited 1.2- to 1.3-fold increases of fermentation capacities, respectively...
April 17, 2018: FEMS Yeast Research
https://www.readbyqxmd.com/read/29657233/reproductive-technologies-for-the-generation-and-maintenance-of-valuable-animal-strains
#19
Takehito Kaneko
Many types of mutant and genetically engineered strains have been produced in various animal species. Their numbers have dramatically increased in recent years, with new strains being rapidly produced using genome editing techniques. In the rat, it has been difficult to produce knockout and knock-in strains because the establishment of stem cells has been insufficient. However, a large number of knockout and knock-in strains can currently be produced using genome editing techniques, including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system...
April 14, 2018: Journal of Reproduction and Development
https://www.readbyqxmd.com/read/29656128/agrobacterium-rhizogenes-mediated-transformation-of-a-dioecious-plant-model-silene-latifolia
#20
Vojtech Hudzieczek, Radim Cegan, Tomas Cermak, Nela Bacovska, Zuzana Machalkova, Karel Dolezal, Lucie Plihalova, Daniel Voytas, Roman Hobza, Boris Vyskot
Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S...
April 12, 2018: New Biotechnology
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