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University of Leicester

7 ARTICLES PUBLISHED IN JoVE

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Biology

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Nicola Portolano 1, Peter J. Watson 1, Louise Fairall 1, Christopher J. Millard 1, Charles P. Milano 1, Yun Song 1, Shaun M. Cowley 1, John W.R. Schwabe 1
1Department of Biochemistry, University of Leicester

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

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Biology

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Thimma R. Reddy 1, Emma J. Kelsall 1, Léna M.S. Fevat 2, Sarah E. Munson *3, Shaun M. Cowley *1
1Department of Biochemistry, University of Leicester, 2Center for Fisheries, Environment and Aquaculture Sciences, 3ES Cell Facility, Centre for Core Biotechnology Services, University of Leicester

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

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Bioengineering

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall
Jack C. Bridge 1, Jonathan W. Aylott 2, Christopher E. Brightling 5, Amir M. Ghaemmaghami 3, Alan J. Knox 4, Mark P. Lewis 6, Felicity R.A.J. Rose 1, Gavin E. Morris 1
1Division of Drug Delivery and Tissue Engineering, University of Nottingham, 2Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, 3Division of Immunology and Allergy, School of Molecular Medical Sciences, University of Nottingham, 4Division of Respiratory Medicine, School of Clinical Sciences, University of Nottingham, 5NIHR Respiratory Biomedical Research Unit, University of Leicester, 6School of Sport, Exercise, and Health Sciences, Loughborough University

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

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Medicine

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice
Melissa Trotman-Lucas 1, Michael E. Kelly 2, Justyna Janus 2, Claire L. Gibson 1,3
1Department of Neuroscience, Psychology and Behaviour, University of Leicester, 2Preclinical Imaging Facility, Core Biotechnology Services, University of Leicester, 3School of Psychology, University of Nottingham

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

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Biology

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry
Elliot Byford 1, Matthew Carr 1, Lucia Piñon 2, Matthew J. Ahearne 1, Simon D. Wagner 1
1Leicester Cancer Research Centre and Ernest and Helen Scott Haematology Research Institute, University of Leicester, 2MRC Toxicology Unit, University of Leicester

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

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Cancer Research

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients
Giuditta Viticchié 1, Ian Powley 1, Constantinos Demetriou 1, James Cooper 1, Michael Butterworth 1, Meeta Patel 1, Naila Abid 1, Gareth Miles 1, Lynne Howells 1, Howard Pringle 1, Marion MacFarlane 2, Catrin Pritchard 1
1Leicester Cancer Research Center, University of Leicester, 2MRC Toxicology Unit

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

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Environment

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance
Ming Hu 1, Zhengjie Liu 1,2, Ziang Song 1,3, Lulu Li 1, Xiaonan Zhao 1, Yanbo Luo 1, Qing Zhang 1, Yibao Chen 1, Xiaohui Xu 1, Yahui Dong 1, Nataliia Hrabchenko 1, Wei Zhang 3, Yuqing Liu 1
1Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, P.R. China, Key Laboratory of Livestock and Poultry Multi-omics of MARA, 2Department of Genetic and Genomes Biology, University of Leicester, 3College of Veterinary Medicine, Nanjing Agricultural University

Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively.

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