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University of Wisconsin, Madison

8 ARTICLES PUBLISHED IN JoVE

Biology

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Katie E. Holmes¹, Matthew J. Wyatt², Yu-chi Shen², Deborah A. Thompson^2,3, Kate F. Barald^2,4

¹Department of Veterinary Science, University of Wisconsin, Madison, ²Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, ³Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, ⁴Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Biology

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Chao Liang^1,2, Harry W. Read², Teri C. Balser^2,3

¹DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, ²Department of Soil Science, University of Wisconsin, Madison, ³Department of Soil and Water Science, University of Florida

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-µCP) and Sequential Nucleophilic Substitution

Gavin T. Knight^*1, Tyler Klann^*1, Jason D. McNulty^1,2, Randolph S. Ashton¹

¹Department of Biomedical Engineering, University of Wisconsin, Madison, ²Department of Mechanical Engineering, University of Wisconsin, Madison

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

Environment

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Lawrence G. Oates¹, Harry W. Read², Jessica L. M. Gutknecht³, David S. Duncan¹, Teri B. Balser⁴, Randall D. Jackson¹

¹Department of Agronomy and Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, ²Department of Soil Science, University of Wisconsin - Madison, ³Department of Soil, Water, and Climate, University of Minnesota, ⁴Faculty of Science and Engineering, Curtin University

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Biology

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Thomas R. Vincent¹, James Canham¹, Masatsugu Toyota^2,3,4, Marieta Avramova¹, Sam T. Mugford⁵, Simon Gilroy², Anthony J. Miller¹, Saskia Hogenhout⁵, Dale Sanders¹

¹Department of Metabolic Biology, John Innes Centre, Norwich Research Park, ²Department of Botany, University of Wisconsin, Madison, ³Department of Biochemistry and Molecular Biology, Saitama University, ⁴Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), ⁵Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

JoVE Journal

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Takuya Uemura¹, Jiaqi Wang¹, Yuri Aratani¹, Simon Gilroy², Masatsugu Toyota^1,2

¹Department of Biochemistry and Molecular Biology, Saitama University, ²Department of Botany, University of Wisconsin

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Biochemistry

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Bryan S. Sibert^*1,2,3, Joseph Y. Kim^*1,4, Jie E. Yang^1,2,3, Elizabeth R. Wright^1,2,3,5

¹Department of Biochemistry, University of Wisconsin, Madison, ²Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, ³Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, ⁴Department of Chemistry, University of Wisconsin, Madison, ⁵Morgridge Institute for Research

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Behavior

A Common Marmoset Model of Mother-Infant Intervention for Breastfeeding Disorders in the Presence of Paternal Inhibition and Maternal Neglect

Mayuko Yoda^1,2, Yoshimasa Kamei³, Hayato Sakurai⁴, Hiroko Kakei⁴, Ting Tao^1,2,4, Hideo Yamanouchi⁴, Tetsuya Kunikata⁴, Masanori Hariyama⁵, Ricki Colman⁶, Mamiko Koshiba^1,4,5

¹Graduate School of Sciences and Technology for Innovation, Yamaguchi University, ²Faculty of Health Sciences and Nursing, Juntendo University, ³Obstetrician, Medical Department, Saitama Medical University, ⁴Department of Pediatrics, Saitama Medical University, ⁵Graduate School of Information Sciences, Tohoku University, ⁶Department of Cell and Regenerative Biology and Wisconsin National Primate Research Center, University of Wisconsin, Madison

Here, we present a nonhuman primate model of mother-infant intervention for breastfeeding disorders in the presence of paternal inhibition and maternal neglect. The mate model video complements education to support primate and human caregivers with respect to infants with breastfeeding problems such as pain.