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University of Leicester

7 ARTICLES PUBLISHED IN JoVE

Biology

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Nicola Portolano¹, Peter J. Watson¹, Louise Fairall¹, Christopher J. Millard¹, Charles P. Milano¹, Yun Song¹, Shaun M. Cowley¹, John W.R. Schwabe¹

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.

Biology

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Thimma R. Reddy¹, Emma J. Kelsall¹, Léna M.S. Fevat², 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.

Bioengineering

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Jack C. Bridge¹, Jonathan W. Aylott², Christopher E. Brightling⁵, Amir M. Ghaemmaghami³, Alan J. Knox⁴, Mark P. Lewis⁶, Felicity R.A.J. Rose¹, Gavin E. Morris¹

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.

Medicine

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

Melissa Trotman-Lucas¹, Michael E. Kelly², Justyna Janus², 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.

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 Byford¹, Matthew Carr¹, Lucia Piñon², Matthew J. Ahearne¹, Simon D. Wagner¹

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.

Cancer Research

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

Giuditta Viticchié¹, Ian Powley¹, Constantinos Demetriou¹, James Cooper¹, Michael Butterworth¹, Meeta Patel¹, Naila Abid¹, Gareth Miles¹, Lynne Howells¹, Howard Pringle¹, Marion MacFarlane², Catrin Pritchard¹

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.

Environment

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

Ming Hu¹, Zhengjie Liu^1,2, Ziang Song^1,3, Lulu Li¹, Xiaonan Zhao¹, Yanbo Luo¹, Qing Zhang¹, Yibao Chen¹, Xiaohui Xu¹, Yahui Dong¹, Nataliia Hrabchenko¹, Wei Zhang³, Yuqing Liu¹

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.