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Pica in Rats as a Preclinical Model of Emesis
T. Gregg Davis
The ability to assess the potential for gastrointestinal adverse events in a preclinical setting is a challenge in the development of new drugs, as the vast majority of in vivo research is conducted in rodent species lacking a vomiting reflex. The use of higher species capable of emesis is often
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Cerebral Cortex Electroporation to Study Projection Neuron Migration
Emilie Pacary, François Guillemot
Brain electroporation is a rapid and powerful approach to study neuronal development. In particular, this technique has become a method of choice for studying the process of radial migration of projection neurons in the embryonic cerebral cortex. This method has considerably helped to describe in
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Cigarette Smoke Extract: A Preclinical Model of Tobacco Dependence
Candice A. Gellner, Daisy D. Reynaga, Frances M. Leslie
Animal models are used to study many human diseases, one of which is tobacco addiction. Most preclinical models use nicotine alone, although there are >7000 constituents present in tobacco smoke. The clinical literature suggests that cigarettes have a strong addictive potential, which is not
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The Neonatal Ventral Hippocampal Lesion (NVHL) Rodent Model of Schizophrenia
Anne Marie Brady
Animal models are crucial to the study of the neurobiological bases of psychiatric disorders, but schizophrenia is a particularly challenging disorder to model given the complexity and heavily verbal nature of its symptoms. This unit describes a developmental surgical rodent model of schizophrenia,
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Automatic Dendritic Spine Quantification from Confocal Data with Neurolucida 360
Dara L. Dickstein, Daniel R. Dickstein, William G. M. Janssen, Patrick R. Hof, Jacob R. Glaser, Alfredo Rodriguez, Nate O'Connor, Paul Angstman, Susan J. Tappan
Determining the density and morphology of dendritic spines is of high biological significance given the role of spines in synaptic plasticity and in neurodegenerative and neuropsychiatric disorders. Precise quantification of spines in three dimensions (3D) is essential for understanding the
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The Movement Tracker: A Flexible System for Automated Movement Analysis in Invertebrate Model Organisms
Laurent Mouchiroud, Vincenzo Sorrentino, Evan G. Williams, Matteo Cornaglia, Michael V. Frochaux, Tao Lin, Amandine A. Nicolet‐dit‐Félix, Gopal Krishnamani, Tarik Ouhmad, Martin A.M. Gijs, Bart Deplancke, Johan Auwerx
Phenotyping strategies in simple model organisms such as D. melanogaster and C. elegans are often broadly limited to growth, aging, and fitness. Recently, a number of physical setups and video tracking software suites have been developed to allow for accurate, quantitative, and high‐throughput
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Flow Analysis and Sorting of Plant Chromosomes
Jan Vrána, Petr Cápal, Hana Šimková, Miroslava Karafiátová, Jana Čížková, Jaroslav Doležel
Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and
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Whole Blood Analysis of Leukocyte‐Platelet Aggregates
Anja J. Gerrits, Andrew L. Frelinger, Alan D. Michelson
In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte‐platelet aggregate formation is initiated primarily through platelet surface expression of P‐selectin (CD62P), following activation‐dependent degranulation
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Automated Measurement of Blood Vessels in Tissues from Microscopy Images
Neil J. Kelly, Nadine Dandachi, Dmitry A. Goncharov, Andressa Z. Pena, Josiah E. Radder, Alyssa D. Gregory, Yen‐Chun Lai, Adriana S. Leme, Mark T. Gladwin, Elena A. Goncharova, Claudette M. St. Croix, Steven D. Shapiro
The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual
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Genetic Risk Scores
Jessica N. Cooke Bailey, Robert P. Igo
The generation of genome‐wide variation data has become commonplace. However, the potential for interpretation and application of these data for clinical assessment of outcomes of interest, and prediction of disease risk, is currently not fully realized. Many common, complex diseases now have
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Analysis of Heritability Using Genome‐Wide Data
Jacob B. Hall, William S. Bush
Most analyses of genome‐wide association data consider each variant independently without considering or adjusting for the genetic background present in the rest of the genome. New approaches to genome analysis use representations of genomic sharing to better account for confounding factors like
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Molecular Diagnosis of Myotonic Dystrophy
Sujata Chakraborty, Matteo Vatta, Linda L. Bachinski, Ralf Krahe, Stephen Dlouhy, Shaochun Bai
Myotonic dystrophy types 1 (DM1) and 2 (DM2) are autosomal dominant, microsatellite repeat expansion disorders that affect muscle function. Myotonic dystrophy type 1 is caused by CTG repeat expansion in the 3′ UTR region of the DMPK gene. Patients with DM2 have expansion of CCTG repeats in intron 1
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COSMIC: High‐Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer
S.A. Forbes, D. Beare, N. Bindal, S. Bamford, S. Ward, C.G. Cole, M. Jia, C. Kok, H. Boutselakis, T. De, Z. Sondka, L. Ponting, R. Stefancsik, B. Harsha, J. Tate, E. Dawson, S. Thompson, H. Jubb, P.J. Campbell
COSMIC (http://cancer.sanger.ac.uk) is an expert‐curated database of somatic mutations in human cancer. Broad and comprehensive in scope, recent releases in 2016 describe over 4 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but
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Pronuclear Injection‐Based Targeted Transgenesis
Samantha L.P. Schilit, Masato Ohtsuka, Rolen M. Quadros, Channabasavaiah B. Gurumurthy
Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy
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High‐Risk Screening of Fabry Disease: Analysis of Fifteen Urinary Methylated and Non‐Methylated Gb3 Isoforms Using Tandem Mass Spectrometry
Mona Abaoui, Michel Boutin, Pamela Lavoie, Christiane Auray‐Blais
Fabry disease is a multisystemic, X‐linked lysosomal storage disorder caused by mutations in the GLA gene, leading to α‐galactosidase A deficiency and resulting in the accumulation of glycosphingolipids in different tissues and biological fluids. Glycosphingolipid biomarkers, such as
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Acylglycine Analysis by Ultra‐Performance Liquid Chromatography‐Tandem Mass Spectrometry (UPLC‐MS/MS)
Judith A. Hobert, Aiping Liu, Marzia Pasquali
Quantitative analysis of urine acylglycines has shown to be a highly sensitive and specific method with proven clinical utility for the diagnosis of several inherited metabolic disorders including: medium chain acyl‐CoA dehydrogenase deficiency, multiple acyl‐CoA dehydrogenase deficiency, short
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Transcriptome Analysis at the Single‐Cell Level Using SMART Technology
Rachel N. Fish, Magnolia Bostick, Alisa Lehman, Andrew Farmer
RNA sequencing (RNA‐seq) is a powerful method for analyzing cell state, with minimal bias, and has broad applications within the biological sciences. However, transcriptome analysis of seemingly homogenous cell populations may in fact overlook significant heterogeneity that can be uncovered at the
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Strand‐Specific Transcriptome Sequencing Using SMART Technology
Magnolia Bostick, Nathalie Bolduc, Alisa Lehman, Andrew Farmer
Next‐generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA‐seq), which is necessary to
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Preparation of Low‐Input and Ligation‐Free ChIP‐seq Libraries Using Template‐Switching Technology
Nathalie Bolduc, Alisa P. Lehman, Andrew Farmer
Chromatin immunoprecipitation (ChIP) followed by high‐throughput sequencing (ChIP‐seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low‐abundance transcription
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Protein Interaction Profile Sequencing (PIP‐seq)
Shawn W. Foley, Brian D. Gregory
Every eukaryotic RNA transcript undergoes extensive post‐transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA‐binding proteins which recognize their target transcript by both its primary sequence and secondary
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Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species
Ana Cecilia Mestre‐Citrinovitz, Adrián Jorge Sestelo, María Belén Ceballos, José Lino Barañao, Patricia Saragüeta
Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult‐to‐find, endangered and giant species like Jaguar ( Panthera onca ), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and
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GILA, a Replacement for the Soft‐Agar Assay that Permits High‐Throughput Drug and Genetic Screens for Cellular Transformation
Benjamin Izar, Asaf Rotem
For the last five decades, measuring the ability of cells to grow in soft agar has served as the gold standard assay for in vitro cellular transformation. Nevertheless, the soft agar colony formation assay is time consuming and ill‐suited for high‐throughput screens. This unit describes an equally
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