National University of Singapore View Institution's Website 46 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. Biology A High-Throughput Platform for Culture and 3D Imaging of Organoids Gianluca Grenci1,2, Florian Dilasser1, Saburnisha Binte Mohamad Raffi1, Marion Marchand1, Mona Suryana1, Geetika Sahni1, Virgile Viasnoff1,3,4, Anne Beghin1,5 1Mechanobiology Institute (MBI), National University of Singapore, 2Biomedical Engineering Department, National University of Singapore, 3Department of Biological Sciences, National University of Singapore, 4IRL 3639 CNRS, 5Department of Microbiology & Immunology, Immunology Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed. Bioengineering Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint Aman Mahajan*1,2, Sugata Hazra*1,2, Aditya Arora1,3, Dhirendra S. Katti1,2 1Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, 2The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, 3Mechanobiology Institute, National University of Singapore Infrapatellar fat pad mesenchymal stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided. 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. Biology Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control Inn Chuan Ng*1, Li Zhang*2, Narelle Nichola Yi Ying Shen3, Yun Ting Soong4, Chan Way Ng5, Phoebe Kang Sheing Koh1, Yan Zhou3, Hanry Yu1,3,4,5,6 1Department of Physiology & The Institute for Digital Medicine (WisDM), National University of Singapore, 2College of Agriculture and Biology, Zhongkai University of Agriculture and Engineering, 3Mechanobiology Institute, National University of Singapore, 4Institute of Bioengineering and Nanotechnology, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering, 6CAMP, Singapore-MIT Alliance for Research and Technology This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes. Immunology and Infection Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection Zhe Zhang Ryan Lew*1, Jing Liu*1, Hsiao Hui Ong1, Vivian Jiayi Tan2, Annika Luukkainen3, Yew Kwang Ong1,4, Mark Thong1,4, Kia Joo Puan5, Vincent Tak Kwong Chow2,6, Kai Sen Tan1,2,6, De Yun Wang1,6 1Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Haartman Institute, Medicum, University of Helsinki, 4Department of Otolaryngology, Head and Neck Surgery, National University Hospital, National University Health System, 5Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), 6NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses. Immunology and Infection Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster Cheng Teng Ng1, Choon Nam Ong2, Liya E. Yu3, Boon Huat Bay4, Gyeong Hun Baeg4 1NUS Environmental Research Institute & Department of Anatomy, National University of Singapore, 2NUS Environmental Research Institute & School of Public Health, National University of Singapore, 3Department of Civil and Environmental Engineering, National University of Singapore, 4Department of Anatomy, National University of Singapore We describe a detailed protocol for evaluating the toxicological profiles of zinc oxide nanoparticles (ZnO NPs) in particular, the type of cell death in human MRC5 lung fibroblasts and ROS formation in the fruit fly Drosophila. Developmental Biology Injecting Gryllus bimaculatus Eggs Samantha K. Barry1, Taro Nakamura2, Yuji Matsuoka3, Christoph Straub4, Hadley W. Horch4, Cassandra G. Extavour5 1Colby College, 2Division of Evolutionary Developmental Biology, National Institute for Basic Biology, 3Department of Biological Sciences, National University of Singapore, 4Department Biology and Department of Neuroscience, Bowdoin College, 5Department of Organismic and Evolutionary Biology and Department of Molecular and Cellular Biology, Harvard University Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation. Immunology and Infection Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex Masanori Asai*1, Yanwen Li*1, Jasmeet Singh Khara1,2, Camilla A. Gladstone1, Brian D. Robertson3, Paul R. Langford*1, Sandra M. Newton*1 1Section of Pediatric Infectious Diseases and Allergy, Department of Medicine, Imperial College London, 2Department of Pharmacy, National University of Singapore, 3MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Immunology and Infection Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis EnJun Yang1, Jacqueline L.Y. Chee1, Suhanya Duraiswamy2, Siyi Chen3, Kristin Lees3, Swaine L. Chen1,3 1Infectious Diseases Group, Genome Institute of Singapore, 2Department of Chemical and Biomolecular Engineering, National University of Singapore, 3Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection. Immunology and Infection Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation Xiang Zhao1, Maryam Hamidinia1, Joanna Ai Ling Choo1, Chien Tei Too1,2, Zi Zong Ho3, Ee Chee Ren4, Antonio Bertoletti3, Paul A. MacAry1,2,5, Keith G. Gould6, Joanna Brzostek1, Nicholas R.J. Gascoigne1,2,5 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, 4Singapore Immunology Network, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 6Department of Immunology, Wright-Fleming Institute, Imperial College London This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments. Immunology and Infection Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries Elijah W. Chen*1, Chyan Ying Ke*2,3, Joanna Brzostek*1, Nicholas R. J. Gascoigne1, Vasily Rybakin1,4 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Singapore Immunology Network, A*STAR, 3Curiox Biosystems, 4Department of Immunobiology, Rega Institute for Medical Research, Katholieke Universiteit (KU) Leuven This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings. Immunology and Infection Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma Guo Hong*1,2, Shuangyi Fan*3, The Phyu3, Priyanka Maheshwari2, Michal Marek Hoppe2, Hoang Mai Phuong2, Sanjay de Mel4, Michelle Poon4, Siok-Bian Ng2,3, Anand D. Jeyasekharan2,4 1Department of Laboratory Medicine, AnSteel Group General Hospital, 2Cancer Science Institute of Singapore, National University of Singapore, 3Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, 4Department of Haematology-Oncology, National University Health System Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections. Immunology and Infection In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells Yi Lu1,2,3, Xin-Yuan Fu3, Yongliang Zhang1,2 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells. Medicine Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators Tuangsit Wataganara1, Arundhati Gosavi2, Katika Nawapun1, Pradip D. Vijayakumar2, Nisarat Phithakwatchara1, Mahesh Choolani2,3, Lin Lin Su2,3, Arijit Biswas2,3, Citra N. Z. Mattar2,3 1Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Siriraj Hospital, 2Department of Obstetrics and Gynaecology, National University Health Systems, 3Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome. Engineering Atmospheric Pressure Fabrication of Large-Sized Single-Layer Rectangular SnSe Flakes Jizhou Jiang1,2, Calvin Pei Yu Wong2,3, Wenjing Zhang1, Andrew Thye Shen Wee2,4 1SZU-NUS Collaborative Innovation Center for Optoelectronic Science & Technology, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, 2Department of Physics, National University of Singapore, 3NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, 4Centre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore A protocol is presented demonstrating a two-step fabrication technique to grow large-sized single-layer rectangular shaped SnSe flakes on low-cost SiO2/Si dielectrics wafers in an atmospheric pressure quartz tube furnace system. Chemistry Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations Ren Wei Toh1, Jie Sheng Li1, Jie Wu1 1Department of Chemistry, National University of Singapore A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented. Bioengineering Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum Abhijeet Patra1, Tao Ding2, Minghui Hong3, Arthur Mark Richards2, Ten It Wong4, Xiaodong Zhou4, Chester Lee Drum2 1NUS Nanoscience and Nanotechnology Initiative, National University of Singapore, 2Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, 3Department of Electrical and Computer Engineering, National University of Singapore, 4Institute of Materials Research Engineering, A*STAR (Agency for Science, Technology and Research) This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff ∼10-400 pg/mL, depending on the assay). Cancer Research Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60 Yanzhou Zhang1, Grace SuShin Chia1, Cheng Yong Tham1, Sudhakar Jha1,2 1Cancer Science Institute of Singapore, National University of Singapore, 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time. Medicine Using Retinal Imaging to Study Dementia Victor T.T. Chan1, Tiffany H.K. Tso1, Fangyao Tang1, Clement Tham1, Vincent Mok2,3,4, Christopher Chen5,6, Tien Y. Wong7,8, Carol Y. Cheung1 1Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, 2Department of Medicine & Therapeutics, The Chinese University of Hong Kong, 3Therese Pei Fong Chow Research Centre for Prevention of Dementia, The Chinese University of Hong Kong, 4Gerald Choa Neuroscience Centre, The Chinese University of Hong Kong, 5Memory Aging and Cognition Centre, National University Health System, 6Department of Pharmacology, National University of Singapore, 7Singapore Eye Research Institute, Singapore National Eye Centre, 8Duke-NUS Medical School, National University of Singapore The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia. Biochemistry Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers Huijuan You*1, Shimin Le*2,3, Hu Chen2,4, Linyan Qin1, Jie Yan2,4,5 1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 2Mechanobiology Institute, National University of Singapore, 3Department of Physics, National University of Singapore, 4Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, 5Center for Bioimaging Sciences, National University of Singapore A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins. Bioengineering Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light Mona Suryana1, Jegan V. Shanmugarajah1, Sivakumar M. Maniam1, Gianluca Grenci1 1Mechanobiology Institute (MBI), National University of Singapore A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described. Chemistry Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures Xuanwei Ong*1, Shashank Gupta*1, Wen-Ya Wu2, Sabyasachi Chakrabortty1, Yinthai Chan1,3 1Department of Chemistry, National University of Singapore, 2Materials Processing and Characterisation Department, A*STAR, Institute of Materials Research and Engineering, 3Ceramics Department, A*STAR, Institute of Materials Research and Engineering A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here. Biology Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass Hieng Chiong Tie1, Bing Chen1, Xiuping Sun1, Li Cheng2,3, Lei Lu1 1School of Biological Sciences, Nanyang Technological University, 2Bioinformatics Institute, 3School of Computing, National University of Singapore The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein. Medicine Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation Rupesh Agrawal*1,2,3, Praveen Kumar Balne*2, Sai Bo Bo Tun2, Yeo Sia Wey2, Neha Khandelwal1, Veluchamy A. Barathi2,4,5 1National Healthcare Group Eye Institute, Tan Tock Seng Hospital, 2Singapore Eye Research Institute (SERI), Singapore National Eye Center, 3School of Material Science and Engineering, Nanyang Technological University, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University Health Systems, National University of Singapore, 5Ophthalmology Academic Clinical Research Program, DUKE-NUS Graduate Medical School Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described. Immunology and Infection A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue Aihua Hou1,2, Tanima Bose3, K. George Chandy3, Louis Tong1,2,4,5 1Singapore Eye Research Insitute, A Member of SingHealth, 2DUKE-National University of Singapore Medical School, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Singapore National Eye Center, 5Yong Loo Lin School of Medicine, National University of Singapore This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later. Cancer Research A Murine Orthotopic Bladder Tumor Model and Tumor Detection System Sin Mun Tham1, Kesavan Esuvaranathan1,2, Ratha Mahendran1 1Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 2Department of Urology, National University Hospital This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth. Developmental Biology Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model Tingsheng Yu1,2, Christoph Winkler1,2 1Department of Biological Sciences, National University of Singapore, 2NUS Centre for Bioimaging Sciences (CBIS), National University of Singapore Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions. Medicine Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses Chi Ching Goh1,2, Jackson LiangYao Li1, David Becker3, Wolfgang Weninger4,5,6, Veronique Angeli2,7, Lai Guan Ng1,2,5,8 1Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Centenary Institute for Cancer Medicine and Cell Biology, 5Discipline of Dermatology, University of Sydney, 6Department of Dermatology, Royal Prince Alfred Hospital, 7LSI Immunology Programme, National University of Singapore, 8School of Biological Sciences, Nanyang Technological University This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury. Biology Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM) Yilin Wang1, Pakorn Kanchanawong2,3 1Department of Biology, South University of Science and Technology of China, Shenzhen, 2Mechanobiology Institute, Singapore, 3Department of Biomedical Engineering, National University of Singapore We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy. Biology Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle Yan Cheng Ng1,2, Liam K. Fisher2, Veena Salim2, Sangho Kim1,2,3, Bumseok Namgung2 1NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 2Department of Biomedical Engineering, National University of Singapore, 3Department of Surgery, National University of Singapore This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study. Genetics Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production Ai-Qun Yu1,2, Nina Pratomo1,2, Tee-Kheang Ng1,2, Hua Ling1,2, Han-Saem Cho1,2, Susanna Su Jan Leong1,2,3, Matthew Wook Chang1,2 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 3Food Science and Chemical Engineering, Singapore Institute of Technology We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production. Medicine A Step by Step Protocol for Subretinal Surgery in Rabbits Sami Al-Nawaiseh1, Fabian Thieltges1, Zengping Liu1,2, Claudine Strack1, Ralf Brinken1, Norbert Braun3, Marc Wolschendorf3, Arvydas Maminishkis5, Nicole Eter4, Boris V. Stanzel1,6 1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient. Bioengineering Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery Raymond Wing Moon Lam1, Sunny Akogwu Abbah2, Wang Ming1, Mathanapriya Naidu1, Felly Ng1, Hu Tao1, James Goh Cho Hong3,4, Kang Ting5, Wong Hee Kit1,4 1Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 2Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, 3Department of Bioengineering, Faculty of Engineering, National University of Singapore, 4Tissue Engineering Program, National University of Singapore, 5Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs. Bioengineering Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding Paula Benny1,2, Cedric Badowski2, E. Birgitte Lane2, Michael Raghunath1,2,3 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2Epithelial Biology Laboratory, Institute of Medical Biology, A*STAR, 3Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct. Bioengineering Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation Jin-Huat Low1, Chen-Hua Yeow1 1Department of Biomedical Engineering, Advanced Robotics Centre, Singapore Institute for Neurotechnology, National University of Singapore This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels. Developmental Biology Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates Geetika Sahni*1, Jun Yuan*1, Yi-Chin Toh1,2 1Department of Biomedical Engineering, National University of Singapore, 2Singapore Institute of Neurotechnology (SINAPSE), National University of Singapore Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns. Immunology and Infection Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging Shruti Shankar1,2, Meredith E.K. Calvert3,4, Joanne Y. Yew1,2,5 1Temasek Life Sciences Laboratory, 2Department of Biological Science, National University of Singapore, 3Bioimaging and Biocomputing Facility, Temasek Life Sciences Laboratory, 4Histology and Light Microscopy Core, Gladstone Institutes, 5Pacific Biosciences Research Center, University of Hawai'i at Mānoa The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone. Developmental Biology Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues Muznah Khan1, Stephan Gasser1,2 1Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days. Immunology and Infection Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases Song Buck Tay1,3, Jeng Yeong Chow2, Maybelle Kho Go1,3, Wen Shan Yew1,3 1Department of Biochemistry, Yong Loo Lin School of Medicine, 2Department of Chemistry, University of Utah, 3Synthetic Biology Research Consortium, National University of Singapore Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption. Bioengineering Polymeric Microneedle Array Fabrication by Photolithography Himanshu Kathuria1, Jaspreet Singh Kochhar1, Michelle Hui Min Fong1, Michinao Hashimoto2, Ciprian Iliescu3, Hanry Yu3,4,5, Lifeng Kang1 1Department of Pharmacy, National University of Singapore, 2Singapore University of Technology and Design, 3Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research (A*STAR), 4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 5Mechanobiology Institute, National University of Singapore Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. Neuroscience Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents Mahesh Shivarama Shetty1,2, Mahima Sharma1,2, Neo Sin Hui1,2, Ananya Dasgupta1,2, Suma Gopinadhan1,2, Sreedharan Sajikumar1,2 1Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2Neurobiology/Aging Programme, Life Sciences Institute, National University of Singapore This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents. Bioengineering Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules Marie Francene A. Cutiongco1, Benjamin Kim Kiat Teo2, Evelyn King Fai Yim1,2,3 1Department of Biomedical Engineering, National University of Singapore, 2Mechanobiology Institute, Singapore, National University of Singapore, 3Department of Surgery, National University of Singapore Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release. Bioengineering Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration Nicholas Agung Kurniawan1, Parthiv Kant Chaudhuri2, Chwee Teck Lim2,3 1Systems Biophysics Department, FOM Institute AMOLF, 2Mechanobiology Institute, National University of Singapore, 3Department of Biomedical Engineering, National University of Singapore The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described. Biology Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation Yufen Goh*1, Melissa J. Fullwood*1,2,3, Huay Mei Poh1, Su Qin Peh1, Chin Thing Ong1, Jingyao Zhang1, Xiaoan Ruan1, Yijun Ruan1,3 1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control. Immunology and Infection Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT Oktay Kirak1, Eva-Maria Frickel1, Gijsbert M. Grotenbreg1,2, Heikyung Suh1, Rudolf Jaenisch1,3, Hidde L. Ploegh1,3 1Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.
