Texas Children's Hospital View Institution's Website 8 articles published in JoVE Genetics In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila J. Michael Harnish*1, Samantha L. Deal*2, Hsiao-Tuan Chao1,3,4,5, Michael F. Wangler1,2,4, Shinya Yamamoto1,2,4,5 1Department of Molecular and Human Genetics, Baylor College of Medicine, 2Program in Developmental Biology, Baylor College of Medicine, 3Department of Pediatrics, Section of Neurology and Developmental Neuroscience, Baylor College of Medicine, 4 The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases. Genetics Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information Julia Wang1,2, Zhandong Liu3,4, Hugo J. Bellen1,4,5,6,7, Shinya Yamamoto1,4,5,6 1Program in Developmental Biology, Baylor College of Medicine, 2Medical Scientist Training Program, Baylor College of Medicine, 3Department of Pediatrics, Baylor College of Medicine, 4 Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts. Genetics Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9 Lorenzo Brunetti*1,2,3, Michael C. Gundry*1,2,4, Ayumi Kitano4, Daisuke Nakada1,2,4, Margaret A. Goodell1,2,4,5 1Stem Cells & Regenerative Medicine Center, Baylor College of Medicine, 2Center for Cell and Gene Therapy, Baylor College of Medicine, 3Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, 4Department of Molecular & Human Genetics, Baylor College of Medicine, 5 A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost. Immunology and Infection Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs Maran L. Sprouse1, Gabriele Blahnik2, Thomas Lee1, Natalie Tully1, Pinaki Benarjee3, Eddie A. James2, Maria J. Redondo4, Matthew L. Bettini1, Maria Bettini1 1 The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression. Immunology and Infection Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer Peilin Zheng*1,2, Grant Bertolet*1,2,3, Yuhui Chen1,2, Shengjian Huang1,2, Dongfang Liu1,2,3 1 We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques. Medicine Implantation of Total Artificial Heart in Congenital Heart Disease Iki Adachi1,2, David S. L. Morales3 1 This is a case report of a patient with congenitally corrected transposition of the great arteries (CCTGA) who received a total artificial heart (TAH) as a bridge to heart transplant. The TAH was successfully implanted with modifications to accommodate the patient's congenitally malformed heart. Neuroscience Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue Kevin Ung1, Benjamin R. Arenkiel1,2,3 1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Department of Neuroscience, Baylor College of Medicine (BCM), 3Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue. Neuroscience A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices Jennifer Selever1, Jian-Qiang Kong2, Benjamin R. Arenkiel3,4 1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Precisionary Instruments Inc., 3Departments of Molecular & Human Genetics and Neuroscience, Baylor College of Medicine (BCM), 4Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.