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University of Dundee

11 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion
Yi-Zhen Ng 1,2, Andrew P. South 1
1Division of Cancer Research, Ninewells Hospital and Medical School, University of Dundee, 2Institute of Medical Biology, A*Star, Singapore

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

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Biology

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
John Biddlestone 1, Jimena Druker 1, Alena Shmakova 1, Gus Ferguson 1, Jason R. Swedlow 1, Sonia Rocha 1
1Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, UK

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

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Biology

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
John S. Bett 1, Adel F. M. Ibrahim 1, Amit K. Garg 1, Sonia Rocha 2, Ronald T. Hay 1,2
1MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, 2Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee

Here, we describe a methodology to perform a targeted siRNA “ubiquitome” screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.  This can be adapted to any biological pathway where a robust read out of reporter activity is available.

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Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture
Wikrom Wongpaiboonwattana 1, Marios P. Stavridis 1
1Division of Cancer Research, College of Medicine, Dentistry and Nursing, University of Dundee

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

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Immunology and Infection

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa
Kushal N. Rugjee 1, Shi-qi An 1, Robert P. Ryan 1
1Division of Molecular Microbiology, College of Life Sciences, University of Dundee

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

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Developmental Biology

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps
Charlotte S.L. Bailey *1, Robert A. Bone *1, Philip J. Murray 2, J. Kim Dale 3
1The Danish Stem Cell Center (DanStem), University of Copenhagen, 2Division of Mathematics, University of Dundee, 3Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

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Developmental Biology

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Charles A. C. Williams 1, Nathanael S. Gray 2,3, Greg M. Findlay 1
1The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2Department of Cancer Biology, Dana-Farber Cancer Institute, 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition.

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Biochemistry

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation
Ying Fan *1, Francesca Tonelli *1, Shalini Padmanabhan 2, Marco A.S. Baptista 2, Lindsey Riley 2, Danielle Smith 3, Connie Marras 4, Andrew Howden 5, Dario R. Alessi 1, Esther Sammler 1,6
1MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2The Michael J. Fox Foundation for Parkinson's Research, 3Department of Medical and Molecular Genetics, Indiana University School of Medicine, 4Morton and Gloria Shulman Movement Disorders Centre and the Edmond J. Safra Program in Parkinson's Disease, Toronto Western Hospital, University of Toronto, 5Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, 6Division of Molecular and Clinical Medicine, School of Medicine, Ninewells Hospital and Medical School, University of Dundee

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

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Developmental Biology

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging
Jens Januschke 1, Nicolas Loyer 1
1Cell & Developmental Biology, School of Life Sciences, University of Dundee

This protocol describes applications of sample immobilization using Fibrin clots, limiting drifting, and allowing the addition and washout of reagents during live imaging. Samples are transferred to a drop of Fibrinogen containing culture medium on the surface of a coverslip, after which polymerization is induced by adding thrombin.

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Medicine

A Stable Phantom Material for Optical and Acoustic Imaging
Lina Hacker 1,2, Aoife M. Ivory 3, James Joseph 4,5, Janek Gröhl 1,2, Bajram Zeqiri 3, Srinath Rajagopal 3, Sarah E. Bohndiek 1,2
1Department of Physics, University of Cambridge, 2Cancer Research UK Cambridge Institute, University of Cambridge, 3Ultrasound and Underwater Acoustics Group, Department of Medical, Marine and Nuclear Physics, National Physical Laboratory, 4School of Science and Engineering, University of Dundee, 5Centre for Medical Engineering and Technology, University of Dundee

This protocol describes the fabrication of a stable, biologically relevant phantom material for optical and acoustic biomedical imaging applications, featuring independently tunable acoustic and optical properties.

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Biology

Galleria mellonella as an Antimicrobial Screening Model
Thomas E. Barton 1, Liberty Duignan 2, Aras Kadioglu 2, Joanne L. Fothergill 2, Daniel R. Neill 1
1Division of Molecular Microbiology, School of Life Sciences, University of Dundee, 2Department of Clinical Infection, Microbiology and Immunology, University of Liverpool

This study presents a standardized framework for optimizing G. mellonella infection models for use in preclinical antimicrobial assessment. The application of a G. mellonella model as part of a preclinical antimicrobial development pipeline could decrease the number of ineffective compounds progressing to clinical trials.

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