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van andel institute

5 ARTICLES PUBLISHED IN JoVE

Biology

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Juan Du^1,2, Hendrik Rietman¹, Vivianne G. A. A. Vleeshouwers¹

¹Wageningen UR Plant Breeding, Wageningen University, ²Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Ministry of Education, National Centre for Vegetable Improvement (Central China), Potato Engineering and Technology Research Centre of Hubei Province, Huazhong Agricultural University

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Cancer Research

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Jinsen Lu¹, Lele Wu², Xiaoke Wang², Jinhang Zhu², Juan Du², Bing Shen²

¹School of Basic Medical Sciences, Anhui Medical University, ²Department of Orthopedics, Anhui Provincial Hospital, Anhui Medical University

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Biochemistry

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Emery Haley^*1, Wooyoung Choi^*1, Chen Fan¹, Weinan Sun^2,3, Juan Du¹, Wei Lü¹

¹Van Andel Institute, ²Vollum Institute, ³Janelia Research Campus

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Bioengineering

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

Chen Fan¹, Lay Keng Priscilla Lim¹, Zihao Wu¹, Bhavya Sharma¹, Shi Qi Gan^1,2, Kun Liang¹, Zee Upton^1,3, David Leavesley¹

¹Skin Research Institute of Singapore, Agency for Science, Technology and Research (A*STAR), ²School of Chemical and Life Sciences, Singapore Polytechnic, ³Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR)

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

Genetics

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Chih-Hsiang Yang^1,2, John Longinotto², Ilaria Panzeri^1,2, Laura Arrigoni², Vanessa Wegert¹, Steffen Heyne², Gabriel Seifert³, Adelheid Lempradl^1,2, Ulrike Bönisch², John Andrew Pospisilik^1,2

¹Van Andel Institute, ²Max Planck Institute of Immunobiology and Epigenetics, ³Department of General and Visceral Surgery, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.