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genetic engineering, neuron

Wei Zhang, Luc Girard, Yu-An Zhang, Tomohiro Haruki, Mahboubeh Papari-Zareei, Victor Stastny, Hans K Ghayee, Karel Pacak, Trudy G Oliver, John D Minna, Adi F Gazdar
Background: Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth. Methods: To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models...
February 2018: Translational Lung Cancer Research
Yongping Chai, Zhiwen Zhu, Guangshuo Ou
During C. elegans larval development, the Q neuroblasts produce their lineage by three rounds of divisions along with continuous cell migrations. Their neuronal progeny is dispersed from the pharynx to the anus. This in vivo system to study cell migration is appealing for several reasons. The lineage development is stereotyped; functional analysis and genomic screens are rendered easy and powerful thanks to powerful tools; transgenic manipulations and genome engineering are efficient and can be conveniently combined with live-cell imaging...
2018: Methods in Molecular Biology
Alessandro Bertero, Loukia Yiangou, Stephanie Brown, Daniel Ortmann, Matthias Pawlowski, Ludovic Vallier
The difficulties involved in conditionally perturbing complex gene expression networks represent major challenges toward defining the mechanisms controlling human development, physiology, and disease. We developed an OPTimized inducible KnockDown (OPTiKD) platform that addresses the limitations of previous approaches by allowing streamlined, tightly-controlled, and potent loss-of-function experiments for both single and multiple genes. The method relies on single-step genetic engineering of the AAVS1 genomic safe harbor with an optimized tetracycline-responsive cassette driving one or more inducible short hairpin RNAs (shRNAs)...
February 28, 2018: Current Protocols in Stem Cell Biology
Mikhail E Sokolov, Farid V Bashirov, Vage A Markosyan, Tatyana V Povysheva, Filip O Fadeev, Andrey A Izmailov, Maxim S Kuztetsov, Zufar Z Safiullov, Maxim M Shmarov, Boris S Naroditskyi, András Palotás, Rustem R Islamov
Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC) transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury...
2018: Frontiers in Pharmacology
Youmei Wu, María José Luna, Lauren S Bonilla, Nicholas J P Ryba, James M Pickel
Design and engineering of complex knockin mice has revolutionized the in vivo manipulation of genetically defined cells. Recently development of the bacterial clustered regularly interspersed short palindromic repeats (CRISPR) associated protein 9 (Cas9) system for single site cleavage of mammalian genomes has opened the way for rapid generation of knockin mice by targeting homology directed repair to selected cleavage sites. We used this approach to generate new lines of mice that will be useful for a variety of aspects of neuroscience research...
2018: PloS One
Matthieu Raveau, Denis Polygalov, Roman Boehringer, Kenji Amano, Kazuhiro Yamakawa, Thomas J McHugh
Down syndrome, the leading genetic cause of intellectual disability, results from an extra-copy of chromosome 21. Mice engineered to model this aneuploidy exhibit Down syndrome-like memory deficits in spatial and contextual tasks. While abnormal neuronal function has been identified in these models, most studies have relied on in vitro measures. Here, using in vivo recording in the Dp(16)1Yey model, we find alterations in the organization of spiking of hippocampal CA1 pyramidal neurons, including deficits in the generation of complex spikes...
February 27, 2018: ELife
Kiryl D Piatkevich, Erica E Jung, Christoph Straub, Changyang Linghu, Demian Park, Ho-Jun Suk, Daniel R Hochbaum, Daniel Goodwin, Eftychios Pnevmatikakis, Nikita Pak, Takashi Kawashima, Chao-Tsung Yang, Jeffrey L Rhoades, Or Shemesh, Shoh Asano, Young-Gyu Yoon, Limor Freifeld, Jessica L Saulnier, Clemens Riegler, Florian Engert, Thom Hughes, Mikhail Drobizhev, Balint Szabo, Misha B Ahrens, Steven W Flavell, Bernardo L Sabatini, Edward S Boyden
We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy...
February 26, 2018: Nature Chemical Biology
Satoshi Iwano, Mayu Sugiyama, Hiroshi Hama, Akiya Watakabe, Naomi Hasegawa, Takahiro Kuchimaru, Kazumasa Z Tanaka, Megumu Takahashi, Yoko Ishida, Junichi Hata, Satoshi Shimozono, Kana Namiki, Takashi Fukano, Masahiro Kiyama, Hideyuki Okano, Shinae Kizaka-Kondoh, Thomas J McHugh, Tetsuo Yamamori, Hiroyuki Hioki, Shojiro Maki, Atsushi Miyawaki
Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized...
February 23, 2018: Science
Rose B Creed, Matthew S Goldberg
Preclinical research on Parkinson's disease has relied heavily on mouse and rat animal models. Initially, PD animal models were generated primarily by chemical neurotoxins that induce acute loss of dopaminergic neurons in the substantia nigra. On the discovery of genetic mutations causally linked to PD, mice were used more than rats to generate laboratory animals bearing PD-linked mutations because mutagenesis was more difficult in rats. Recent advances in technology for mammalian genome engineering and optimization of viral expression vectors have increased the use of genetic rat models of PD...
February 8, 2018: Movement Disorders: Official Journal of the Movement Disorder Society
Stefano Pietropaoli, Alessia Leonetti, Chiara Cervetto, Arianna Venturini, Roberta Mastrantonio, Giulia Baroli, Tiziana Persichini, Marco Colasanti, Guido Maura, Manuela Marcoli, Paolo Mariottini, Manuela Cervelli
Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify new potential proteins that could represent key factors in excitotoxic stress and to study the relationship between polyamine catabolism and excitotoxic damage, a novel transgenic mouse line overexpressing spermine oxidase enzyme in the neocortex (Dach-SMOX) has been engineered. These transgenic mice are more susceptible to excitotoxic injury and display a higher oxidative stress, highlighted by 8-Oxo-2'-deoxyguanosine increase and activation of defense mechanisms, as demonstrated by the increase of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the nucleus...
February 3, 2018: Molecular Neurobiology
Albert Ruzo, Gist F Croft, Jakob J Metzger, Szilvia Galgoczi, Lauren J Gerber, Cecilia Pellegrini, Hanbin Wang, Maria Fenner, Stephanie Tse, Adam Marks, Corbyn Nchako, Ali H Brivanlou
Huntington's disease (HD) is a fatal neurodegenerative disease caused by expansion of CAG repeats in the Huntingtin gene (HTT). Neither its pathogenic mechanisms nor the normal functions of HTT are well understood. To model HD in humans, we engineered a genetic allelic series of isogenic human embryonic stem cell (hESC) lines with graded increases in CAG repeat length. Neural differentiation of these lines unveiled a novel developmental HD phenotype: the appearance of giant multinucleated telencephalic neurons at an abundance directly proportional to CAG repeat length, generated by a chromosomal instability and failed cytokinesis over multiple rounds of DNA replication...
January 29, 2018: Development
Edison Chen, Valeria Lallai, Yasmine Sherafat, Nickolas P Grimes, Anna N Pushkin, J P Fowler, Christie D Fowler
The recent development of transgenic rodent lines expressing cre recombinase in a cell-specific manner, along with advances in engineered viral vectors, has permitted in-depth investigations into circuit function. However, emerging evidence has begun to suggest that genetic modifications may introduce unexpected caveats. In the current studies, we sought to extensively characterize male and female mice from both the ChAT(BAC)-Cre mouse line, created with the bacterial artificial chromosome (BAC) method, and ChAT(IRES)-Cre mouse line, generated with the internal ribosome entry site (IRES) method...
January 25, 2018: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Noboru Hiroi
Copy number variants are deletions and duplications of a few thousand to million base pairs and are associated with extraordinarily high levels of autism spectrum disorder, schizophrenia, intellectual disability or attention-deficit/hyperactivity disorder. The unprecedented levels of robust and reproducible penetrance of copy number variants make them one of the most promising and reliable entry points to delve into the mechanistic bases of many mental disorders. However, the precise mechanistic bases of these associations still remain elusive in humans due to the many genes encoded in each copy number variant and the diverse associated phenotypic features...
January 25, 2018: Psychiatry and Clinical Neurosciences
Alessandro Soloperto, Anna Boccaccio, Andrea Contestabile, Monica Moroni, Grace I Hallinan, Gemma Palazzolo, John Chad, Katrin Deinhardt, Dario Carugo, Francesco Difato
Development of remote stimulation techniques for neuronal tissues represents a challenging goal. Among the potential methods, mechanical stimuli are the most promising vector to convey information non-invasively into intact brain tissue. In this context, selective mechano-sensitization of neuronal circuits would pave the way to develop a new cell-type specific stimulation approach. We report here for the first time the development and characterization of mechano-sensitized neuronal networks through the heterologous expression of an engineered bacterial large conductance mechanosensitive ion channel (MscL)...
January 19, 2018: Journal of Cell Science
Bradford E Hall, Michaela Prochazkova, Matthew R Sapio, Paul Minetos, Natalya Kurochkina, B K Binukumar, Niranjana D Amin, Anita Terse, John Joseph, Stephen J Raithel, Andrew J Mannes, Harish C Pant, Man-Kyo Chung, Michael J Iadarola, Ashok B Kulkarni
Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5...
January 19, 2018: Scientific Reports
Yi Yang, Han Wu, Xiangjin Kang, Yanhui Liang, Ting Lan, Tianjie Li, Tao Tan, Jiangyun Peng, Quanjun Zhang, Geng An, Yali Liu, Qian Yu, Zhenglai Ma, Ying Lian, Boon Seng Soh, Qingfeng Chen, Ping Liu, Yaoyong Chen, Xiaofang Sun, Rong Li, Xiumei Zhen, Ping Liu, Yang Yu, Xiaoping Li, Yong Fan
Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m...
January 9, 2018: Protein & Cell
Hélène Martin-Yken, Camille Gironde, Sylvain Derick, Hélène Taiana Darius, Christophe Furger, Dominique Laurent, Mireille Chinain
Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms...
January 4, 2018: Environmental Research
Eiji Shigetomi, Yukiho J Hirayama, Kazuhiro Ikenaka, Kenji F Tanaka, Schuichi Koizumi
Fine processes of astrocytes enwrap synapses and are well-positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+ signals. Astrocytes display numerous spontaneous microdomain Ca2+ signals; however, it is not clear if such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask if GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice...
January 5, 2018: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Estela de O Lima, Tatiane M Guerreiro, Carlos Fernando O R Melo, Diogo N de Oliveira, Daisy Machado, Marcelo Lancelloti, Rodrigo R Catharino
Recently, microcephaly cases have increased in Americas and have been matter of concern due to Zika virus (ZIKV) recent outbreak. Previous studies have shown that ZIKV-infected progenitor neuronal cells present morphological abnormalities and increased rates of cell death, which may be indicators of microcephaly causes. As recent studies indicate Zika virus' tropism for brain cells, how would a glioblastoma (GBM) lineage behave under ZIKV infection, considering GBM the most common and malignant brain tumor in adults, presenting extreme chemoresistance and high morbidity and mortality rates? The current trend of using genetically engineered oncolytic pathogens as a safe way to eliminate tumors is under development, with trials already in course...
December 28, 2017: Journal of Mass Spectrometry: JMS
Zhijie Chen, Tan M Truong, Hui-Wang Ai
Fluorescent protein-based biosensors are indispensable molecular tools for life science research. The invention and development of high-fidelity biosensors for a particular molecule or molecular event often catalyze important scientific breakthroughs. Understanding the structural and functional organization of brain activities remain a subject for which optical sensors are in desperate need and of growing interest. Here, we review genetically encoded fluorescent sensors for imaging neuronal activities with a focus on the design principles and optimizations of various sensors...
2017: Chemosensors (Basel, Switzerland)
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