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Rutgers, the State University of New Jersey

14 ARTICLES PUBLISHED IN JoVE

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Biology

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment
Paula Stein 1, Karen Schindler 1
1Department of Biology, University of Pennsylvania

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

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Neuroscience

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals
David J. Barker 1, David H. Root 2, Kevin R. Coffey 1, Sisi Ma 1, Mark O. West 1
1Department of Psychology, Rutgers, the State University of New Jersey, 2Neuronal Networks Section, Integrative Neuroscience Research Branch, National Institute on Drug Abuse

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges. Methods for performing this technique and the equipment necessary are described. Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

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Neuroscience

Building An Open-source Robotic Stereotaxic Instrument
Kevin R. Coffey 1, David J. Barker 1, Sisi Ma 1, Mark O. West 1
1Department of Psychology, Rutgers, The State University of New Jersey

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

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Immunology and Infection

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH
Sarah M. Misenko 1, Samuel F. Bunting 1
1Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

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Biology

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System
Ying Zhang 1, Juan Dong 1,2
1The Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, 2The Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

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Immunology and Infection

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions
Jansy P. Sarathy 1, Hsin-pin Ho Liang 1, Danielle Weiner 2, Jacqueline Gonzales 2, Laura E. Via 2, Véronique Dartois 1
1Public Health Research Institute Centre, New Jersey Medical School, Rutgers, 2Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

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JoVE Core

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)
Matthew Zimmerman 1, Landry Blanc 1, Pei-Yu Chen 1, Véronique Dartois 1, Brendan Prideaux 1
1Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

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Education

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I
Diego Marin *1,2, Alexandra L. Nguyen *3, Richard T. Scott, Jr. 1,2, Karen Schindler 3
1IVI-RMA New Jersey, 2Jefferson College of Biomedical Sciences, Thomas Jefferson University, 3Department of Genetics, Rutgers, The State University of New Jersey

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

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Biology

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography
Ying Zhang *1, Liming Ma *1, Xiaodi Pan *1, Jinglu Wang 1, Xinyu Guo 1, Jianjun Du 1
1Beijing Key Lab of Digital Plant, Beijing Research Center for Information Technology in Agriculture, Beijing Academy of Agriculture and Forestry Sciences

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

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Immunology and Infection

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria
Shamba Gupta 1, G. Marcela Rodriguez 1
1Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

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JoVE Core

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model
Pei-Yu Chen 1, Lingfeng Qin 2, Michael Simons 1,3
1Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, 2Department of Surgery, Yale University School of Medicine, 3Department of Cell Biology, Yale University School of Medicine

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

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Behavior

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice
Andrew Dieterich 1,2, Christine N. Yohn 1, Benjamin Adam Samuels 1,2
1Department of Psychology, Behavioral and Systems Neuroscience Area, The State University of New Jersey, 2Graduate Program in Neuroscience, Rutgers, The State University of New Jersey

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

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Bioengineering

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection
Joseph J. Sherba 1, Maria Atzampou 1, Hao Lin 2, Jerry W. Shan 2, David I. Shreiber 1, Jeffrey D. Zahn 1
1The Department of Biomedical Engineering, Rutgers, The State University of New Jersey, 2The Department of Mechanical and Aerospace Engineering, Rutgers, The State University of New Jersey

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

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Developmental Biology

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes
Mansour Aboelenain 1,2,3, Karen Schindler 1,2, Cecilia S. Blengini 1,2
1Department of Genetics, Rutgers, The State University of New Jersey, 2Human Genetics Institute of New Jersey, 3Department of Theriogenology, Faculty of Veterinary Medicine, Mansoura University

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

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