The Rockefeller University 8 articles published in JoVE Biology Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory Ana-Maria Tanasescu1 1Laboratory of Membrane Biology and Biophysics, The Rockefeller University This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination. Immunology and Infection Gene Editing of Primary Rhesus Macaque B Cells Harald Hartweger1, Rajeev Gautam2, Yoshiaki Nishimura2, Fabian Schmidt3,5, Kai-Hui Yao1, Amelia Escolano1,6, Mila Jankovic1, Malcolm A. Martin2, Michel C. Nussenzweig1,4 1Laboratory of Molecular Immunology, The Rockefeller University, 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Laboratory of Retrovirology, The Rockefeller University, 4Howard Hughes Medical Institute, The Rockefeller University, 5Laboratory of Applied Virology and Precision Medicine, King Abdullah University of Science and Technology (KAUST), 6Vaccine and Immunotherapy Center, Wistar Institute We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies. Immunology and Infection Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes Veronica Jové*1, Krithika Venkataraman*1, Thomas M. Gabel2, Laura B. Duvall2 1Laboratory of Neurogenetics and Behavior, The Rockefeller University, 2Department of Biological Sciences, Columbia University The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested. Genetics High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology Laetitia Chauve*1, Jérémie Le Pen*2,3,5, Francesca Hodge1, Pia Todtenhaupt1, Laura Biggins1, Eric A. Miska2,3,4, Simon Andrews1, Olivia Casanueva1 1Babraham Institute, 2Gurdon Institute, University of Cambridge, 3Department of Genetics, University of Cambridge, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute, 5Laboratory of Virology and Infectious Disease, The Rockefeller University In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms. Neuroscience Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors Maria G. Garone1, Valeria de Turris2, Alessandro Soloperto2, Carlo Brighi2, Riccardo De Santis3, Francesca Pagani2,4, Silvia Di Angelantonio2,4, Alessandro Rosa1,2 1Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Italy, 2Center for Life Nano Science, Istituto Italiano di Tecnologia, Italy, 3Laboratory of Stem Cell biology and Molecular Embryology, The Rockefeller University, USA, 4Department of Physiology and Pharmacology, Sapienza University of Rome, Italy This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors. Biology The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells Pierre Dourlen1,2, Clemence Levet1, Alexandre Mejat1, Alexis Gambis3, Bertrand Mollereau1 1Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, 2INSERM U744, Institut Pasteur de Lille, Université Lille-Nord de France, 3Howard Hughes Medical Institute, Laboratory of Apoptosis and Cancer, The Rockefeller University The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development. Biology Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors Saranga Naganathan1, Amy Grunbeck1, He Tian1, Thomas Huber1, Thomas P. Sakmar1 1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe. Biology Gavaging Adult Zebrafish Chereen Collymore1,2, Skye Rasmussen2, Ravi J. Tolwani2 1Tri-Institutional Training Program in Laboratory Animal Medicine and Science Memorial Sloan-Kettering Cancer Center, Weill Cornell Medical College, The Rockefeller University, 2Comparative Bioscience Center, The Rockefeller University The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.