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Genetic engineering

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https://www.readbyqxmd.com/read/29786902/engineered-metalloenzymes-with-non-canonical-coordination-environments
#1
Takahiro Hayashi, Donald Hilvert, Anthony P Green
Nature employs a limited number of genetically encoded, metal coordinating residues to create metalloenzymes with diverse structures and functions. Engineered components of the cellular translation machinery can now be exploited to genetically encode non-canonical ligands with user-defined electronic and structural properties. This ability to efficiently install 'chemically programmed' ligands into proteins can provide powerful chemical probes of metalloenzyme mechanisms and presents excellent opportunities to create metalloprotein catalysts with augmented properties and novel activities...
May 22, 2018: Chemistry: a European Journal
https://www.readbyqxmd.com/read/29786899/improved-stable-indocyanine-green-icg-mediated-cancer-optotheranostics-with-naturalized-hepatitis-b-core-particles
#2
Wenjun Shan, Ronghe Chen, Qiang Zhang, Jie Zhao, Binbin Chen, Xi Zhou, Shefang Ye, Shengli Bi, Liming Nie, Lei Ren
In recent years, hepatitis B core protein virus-like particle (HBc VLP) is an impressive biomaterial, which has attracted considerable attention due to favorable properties such as structural stability, high uptake efficiency, and biocompatibility in biomedical applications. Heretofore, only a few attempts have been made to apply it in physical, chemical, and biological therapy for cancer. In this study, a tumor-targeting RGD-HBc VLP is first fabricated through genetic engineering. For image-guided cancer phototherapy, indocyanine green (ICG) is loaded into RGD-HBc VLP via a disassembly/reassembly pathway and electrostatic attraction with high efficiency...
May 22, 2018: Advanced Materials
https://www.readbyqxmd.com/read/29786794/protocols-for-studies-on-genetically-engineered-mouse-models-in-prostate-cancer
#3
Chris W D Armstrong, Oksana Lyubomska, Melissa J LaBonte, David J J Waugh
Cancer studies have entered an era that is heavily focused on the contribution of the tumor microenvironment. For this reason, in vivo experimentation in an immunodeficient model system is no longer fit for purpose. As a consequence, numerous genetically engineered mouse models (GEMMs) which self-develop tumors have been developed to allow experiments to be performed in a fully immunocompetent setting. One of the most commonly used technologies is Cre-loxP recombination due to its unique ability to control target gene expression in a specified tissue type...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29786504/transgenic-pigs-to-the-rescue
#4
Björn Petersen
Genetically engineered pigs that digest their food better could help to reduce phosphorus and nitrogen pollution.
May 22, 2018: ELife
https://www.readbyqxmd.com/read/29786090/crispr-off-target-analysis-in-genetically-engineered-rats-and-mice
#5
Keith R Anderson, Maximilian Haeussler, Colin Watanabe, Vasantharajan Janakiraman, Jessica Lund, Zora Modrusan, Jeremy Stinson, Qixin Bei, Andrew Buechler, Charles Yu, Sobha R Thamminana, Lucinda Tam, Michael-Anne Sowick, Tuija Alcantar, Natasha O'Neil, Jinjie Li, Linda Ta, Lisa Lima, Merone Roose-Girma, Xin Rairdan, Steffen Durinck, Søren Warming
Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq...
May 21, 2018: Nature Methods
https://www.readbyqxmd.com/read/29784537/artificial-intelligence-a-joint-narrative-on-potential-use-in-pediatric-stem-and-immune-cell-therapies-and-regenerative-medicine
#6
REVIEW
Irena Sniecinski, Jerard Seghatchian
Artificial Intelligence (AI) reflects the intelligence exhibited by machines and software. It is a highly desirable academic field of many current fields of studies. Leading AI researchers describe the field as "the study and design of intelligent agents". McCarthy invented this term in 1955 and defined it as "the science and engineering of making intelligent machines". The central goals of AI research are reasoning, knowledge, planning, learning, natural language processing (communication), perception and the ability to move and manipulate objects...
May 9, 2018: Transfusion and Apheresis Science
https://www.readbyqxmd.com/read/29782151/a-computational-workflow-for-the-automated-generation-of-models-of-genetic-designs
#7
Göksel Mısırlı, Tramy Nguyen, James Alastair McLaughlin, Prashant Vaidyanathan, Timothy Jones, Douglas Densmore, Chris J Myers, Anil Wipat
Computational models are essential to engineer predictable biological systems and to scale up this process for complex systems. Computational modelling often requires expert knowledge and data to build models. Clearly, manual creation of models is not scalable for large designs. Despite several automated model construction approaches, computational methodologies to bridge knowledge in design repositories and the process of creating computational models has still not been established. This paper describes a workflow for automatic generation of computational models of genetic circuits from data stored in design repositories using existing standards...
May 21, 2018: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29782018/novel-method-of-plasmid-dna-delivery-to-mouse-bladder-urothelium-by-electroporation
#8
Chuan Yu, Ofir Stefanson, Yueli Liu, Zhu A Wang
Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs...
May 3, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29781340/graphene-oxide-enhances-alginate-encapsulated-cells-viability-and-functionality-while-not-affecting-the-foreign-body-response
#9
Jesús Ciriza, Laura Saenz Del Burgo, Haritz Gurruchaga, Francesc E Borras, Marcella Franquesa, Gorka Orive, Rosa Maria Hernández, José Luis Pedraz
The combination of protein-coated graphene oxide (GO) and microencapsulation technology has moved a step forward in the challenge of improving long-term alginate encapsulated cell survival and sustainable therapeutic protein release, bringing closer its translation from bench to the clinic. Although this new approach in cell microencapsulation represents a great promise for long-term drug delivery, previous studies have been performed only with encapsulated murine C2 C12 myoblasts genetically engineered to secrete murine erythropoietin (C2 C12 -EPO) within 160 µm diameter hybrid alginate protein-coated GO microcapsules implanted into syngeneic mice...
November 2018: Drug Delivery
https://www.readbyqxmd.com/read/29781251/de-novo-design-of-recombinant-spider-silk-proteins-for-material-applications
#10
REVIEW
Ke Zheng, Shengjie Ling
Spider silks are well known for their superior mechanical properties that are stronger and tougher than steel despite being assembled at close to ambient conditions and using water as the solvent. However, it is a significant challenge to utilize spider silks for practical applications due to their limited sources. Fortunately, genetic engineering techniques offer a promising approach to produce useable amounts of spider silk variants. Starting from these recombinant spider silk proteins, a series of experiments and simulations strategies were developed to improve the recombinant spider silk proteins (RSSP) material design and fabrication with the aim of biomimicking the structure-property-function relationships of spider silks...
May 21, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29780583/exploring-local-solvation-environments-of-a-heme-protein-using-the-spectroscopic-reporter-4-cyano-l-phenylalanine
#11
Caroline Kearney, Lukasz T Olenginski, Trexler D Hirn, Gwendolyn D Fowler, Daniyal Tariq, Scott H Brewer, Christine M Phillips-Piro
The vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein Caldanaerobacter subterraneus H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in E. coli . The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O2 ligated and unligated states was confirmed with UV-Vis spectroscopy...
April 9, 2018: RSC Advances
https://www.readbyqxmd.com/read/29780558/long-lived-protein-expression-in-hydrogel-particles-towards-artificial-cells
#12
Xiaoyu Zhou, Han Wu, Miao Cui, Sze Nga Lai, Bo Zheng
Herein we report a new type of cell-mimic particle capable of long-lived protein expression. We constructed the cell-mimic particles by immobilizing the proteinaceous factors of the cell-free transcription and translation system on the polymer backbone of hydrogel particles and encapsulating the plasmid template and ribosome inside the hydrogel. With the continuous supply of nutrients and energy, the protein expression in the cell-mimic particles remained stable for at least 11 days. We achieved the regulation of protein expression in the cell-mimic particles by the usage of lac operon...
May 14, 2018: Chemical Science
https://www.readbyqxmd.com/read/29779578/mouse-models-for-the-analysis-of-gonadotropin-secretion-and-action
#13
REVIEW
Sara Babcock Gilbert, Allyson K Roof, T Rajendra Kumar
Gonadotropins are pituitary gonadotrope-derived glycoprotein hormones. They act by binding to G-protein coupled receptors on gonads. Gonadotropins play critical roles in reproduction by regulating both gametogenesis and steroidogenesis. Although biochemical and physiological studies provided a wealth of knowledge, gene manipulation techniques using novel mouse models gave new insights into gonadotropin synthesis, secretion and action. Both gain of function and loss of function mouse models for understanding gonadotropin action in a whole animal context have already been generated...
June 2018: Best Practice & Research. Clinical Endocrinology & Metabolism
https://www.readbyqxmd.com/read/29778216/nitric-oxide-signalling-in-yeast
#14
Rika I Astuti, Ryo Nasuno, Hiroshi Takagi
Nitric oxide (NO) is a cellular signalling molecule widely conserved among organisms, including microorganisms such as bacteria, yeasts, and fungi, and higher eukaryotes such as plants and mammals. NO is mainly produced by the activities of NO synthase (NOS) or nitrite reductase (NIR). There are several NO detoxification systems, including NO dioxygenase (NOD) and S-nitrosoglutathione reductase (GSNOR). NO homeostasis, based on the balance between NO synthesis and degradation, is important for regulating its physiological functions, since an excess of NO causes nitrosative stress due to the high reactivity of NO and NO-derived compounds...
2018: Advances in Microbial Physiology
https://www.readbyqxmd.com/read/29777593/degradation-of-recombinant-proteins-by-cho-host-cell-proteases-is-prevented-by-matriptase-1-knock-out
#15
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/29777358/generation-of-genetically-engineered-animals-using-engineered-endonucleases
#16
REVIEW
Jong Geol Lee, Young Hoon Sung, In-Jeoung Baek
The key to successful drug discovery and development is to find the most suitable animal model of human diseases for the preclinical studies. The recent emergence of engineered endonucleases is allowing for efficient and precise genome editing, which can be used to develop potentially useful animal models for human diseases. In particular, zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat systems are revolutionizing the generation of diverse genetically-engineered experimental animals including mice, rats, rabbits, dogs, pigs, and even non-human primates that are commonly used for preclinical studies of the drug discovery...
May 17, 2018: Archives of Pharmacal Research
https://www.readbyqxmd.com/read/29777103/bacterial-encapsulins-as-orthogonal-compartments-for-mammalian-cell-engineering
#17
Felix Sigmund, Christoph Massner, Philipp Erdmann, Anja Stelzl, Hannes Rolbieski, Mitul Desai, Sarah Bricault, Tobias P Wörner, Joost Snijder, Arie Geerlof, Helmut Fuchs, Martin Hrabĕ de Angelis, Albert J R Heck, Alan Jasanoff, Vasilis Ntziachristos, Jürgen Plitzko, Gil G Westmeyer
We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT)...
May 18, 2018: Nature Communications
https://www.readbyqxmd.com/read/29776931/shuffling-the-neutral-drift-of-unspecific-peroxygenase-in-saccharomyces-cerevisiae
#18
Javier Martin-Diaz, Carmen Paret, Eva García-Ruiz, Patricia Molina-Espeja, Miguel Alcalde
Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded and active UPO variants were generated...
May 18, 2018: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29776924/tools-for-rapid-genetic-engineering-of-vibrio-fischeri
#19
Karen L Visick, Kelsey M Hodge-Hanson, Alice H Tischler, Allison K Bennett, Vincent Mastrodomenico
Vibrio fischeri is used as a model for number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multi-step process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR SOEing (Splicing by Overlap Extension) with antibiotic cassettes flanked by standardized linker sequences...
May 18, 2018: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29776730/development-of-human-derived-hemoglobin-albumin-microspheres-as-oxygen-carriers-using-shirasu-porous-glass-membrane-emulsification
#20
Seiichi Ohta, Kenichiro Hashimoto, Xiaoting Fu, Masamichi Kamihira, Yasuyuki Sakai, Taichi Ito
Novel hemoglobin-based artificial oxygen carriers are necessary in tissue engineering. We developed human hemoglobin (hHb) and albumin (HSA)-based microspheres using Shirasu porous glass (SPG) membrane emulsification. The obtained microspheres had a uniform size with an average diameter of 15.1 μm measured by optical microscope, which is similar to the diameter of human red blood cells (7-8 μm). The loading amount of hHb in the microspheres was 20 wt%, which is similar to that of red blood cells (33 wt%)...
May 15, 2018: Journal of Bioscience and Bioengineering
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