Duke-NUS Medical School 6 articles published in JoVE Medicine Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice Sai Bo Bo Tun*1, Edwin Shepherdson*2, Hwee Goon Tay2,3, Veluchamy Amutha Barathi1,3,4 1Singapore National Eye Centre, Singapore Eye Research Institute, 2Centre for Vision Research, Duke-NUS Medical School, 3Ophthalmology and Visual Sciences Academic Clinical Program, DUKE-NUS Medical School, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice. Bioengineering Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy Su Yin Chaw1,2, Tina Tzee Ling Wong3,4, Subbu Venkatraman2,5, Ann-Marie Chacko1 1Laboratory for Translational and Molecular Imaging, Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, 2School of Materials Science and Engineering, Nanyang Technological University, 3Singapore National Eye Centre, 4Singapore Eye Research Institute, 5Material Science & Engineering, National University of Singapore We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection. Medicine Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases Ivan Seah*1,2, Zengping Liu*1,3,4, Daniel Soo Lin Wong1, Wendy Wong2, Graham E. Holder1,2,5, Veluchamy Amutha Barathi1,4,6, Gopal Lingam1,2,4, Xinyi Su1,2,3,4, Boris V. Stanzel1,7,8 1Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Ophthalmology, National University Hospital, Singapore, 3Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 4Singapore Eye Research Institute (SERI), 5UCL Institute of Ophthalmology, 6Academic Clinical Program in Ophthalmology, Duke-NUS Medical School, 7Macula Center Saar, Eye Clinic Sulzbach, Knappschaft Hospital Saar, 8Department of Ophthalmology, University of Bonn The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation. Cancer Research Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods Jiajun Yap1, Jimin Yuan1, Zi Heng Tee1, Xuchao Huang1, Wan Hwa Ng1, Jiancheng Hu1,2 1The Laboratory of Cancer Signaling, Division of Cellular and Molecular Research, National Cancer Centre Singapore, 2The Cancer and Stem Cell Program, Duke-NUS Medical School In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay. Genetics Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format Muhammad Khairul Ramlee1, Jing Wang1, Alice M. S. Cheung1, Shang Li1 1Cancer & Stem Cell Biology Programme, Duke-NUS Medical School The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods. Biology Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture Qianfan Bai*1, Zhiqiang Bai*1, Shaohai Xu3, Lei Sun1,2 1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 2Institute of Molecular and Cell Biology, Singapore, 3Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang Technological University An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.
