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Columbia University

12 ARTICLES PUBLISHED IN JoVE

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Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)
Neela Zareen 1, Lloyd A. Greene 2
1Department of Biology, Columbia University, 2Department of Pathology and Cell Biology, Columbia University

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

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Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
Hae Young Lee 1, Lloyd A. Greene 2, Carol A. Mason 2,3, M. Chiara Manzini 2,4
1Department of Genetics and Development, Columbia University , 2Department of Pathology and Cell Biology, Columbia University , 3Department of Neuroscience, Columbia University , 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

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Biology

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
Jessica M. Skeie 1,2, Stephen H. Tsang 3, Vinit B. Mahajan 1,2
1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

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Medicine

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice
Jacqueline J. Glascock 1, Erkan Y. Osman 1, Tristan H. Coady 2, Ferrill F. Rose 1, Monir Shababi 3, Christian L. Lorson 3
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University , 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

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Medicine

Mouse Eye Enucleation for Remote High-throughput Phenotyping
Vinit B. Mahajan 1,2, Jessica M. Skeie 1,2, Amir H. Assefnia 2,3, MaryAnn Mahajan 1,2, Stephen H. Tsang 2,4
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

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Immunology and Infection

Production and Titering of Recombinant Adeno-associated Viral Vectors
Christina McClure *1, Katy L. H. Cole *1, Peer Wulff 1, Matthias Klugmann 2, Andrew J. Murray 1,3
1School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, 2Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, 3Department of Biochemistry and Molecular Biophysics, Columbia University

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

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cobas Screening and Improved Cervical Cancer Screening and Prevention
Jane S. Gibson 1, Ricki Pollycove 2, Thomas C. Wright 3
1Department of Clinical Sciences College of Medicine, University of Central Florida, 2Division of Gynecology, California Pacific Medical Center, 3Department of Pathology and Cell Biology, Columbia University

In order to reduce cervical cancer incidence we need to increase the sensitivity of methods used for screening. Testing for "high-risk" types of HPV greatly improves sensitivity compared to cervical cytology, but is generally thought to reduce specificity. Incorporating genotyping for HPV 16 and 18 together with "high-risk" HPV testing reduces the impact of lower specificity.

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Medicine

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Katherine J. Wert 1,2, Jessica M. Skeie 3,4, Richard J. Davis 1, Stephen H. Tsang 1,3, Vinit B. Mahajan 3,4
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University , 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University , 3Omics Laboratory, University of Iowa , 4Department of Ophthalmology and Visual Sciences, University of Iowa

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

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Biology

Analyzing and Building Nucleic Acid Structures with 3DNA
Andrew V. Colasanti 1, Xiang-Jun Lu 2, Wilma K. Olson 1
1Department of Chemistry & Chemical Biology and BioMaPS Institute for Quantitative Biology, Rutgers - The State University of New Jersey, 2Department of Biological Sciences, Columbia University

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

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Neuroscience

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
Perrine Inquimbert 1, Martin Moll 2, Tatsuro Kohno 3, Joachim Scholz 2
1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University , 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

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Neuroscience

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes
Cuiping Zhao 1, Nathan C. Boles 1, Justine D. Miller 1, Suzanne Kawola 1, Sally Temple 1, Richard J. Davis *1, Jeffrey H. Stern *1
1Neural Stem Cell Institute

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

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Biochemistry

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis
Thiago Cabral *1,2,7,8, Marcus A. Toral *3,4, Gabriel Velez 3,4, James E. DiCarlo 1,2, Anuradha M. Gore 3, MaryAnn Mahajan 3, Stephen H. Tsang 1,2, Alexander G. Bassuk 5,6, Vinit B. Mahajan 3,9
1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

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