The Rockefeller University

7 articles published in JoVE

• Immunology and Infection

  
  Gene Editing of Primary Rhesus Macaque B Cells Harald Hartweger¹, Rajeev Gautam², Yoshiaki Nishimura², Fabian Schmidt^3,5, Kai-Hui Yao¹, Amelia Escolano^1,6, Mila Jankovic¹, Malcolm A. Martin², Michel C. Nussenzweig^{1,4 1}Laboratory 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. Duvall^{2 1}Laboratory 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. Miska^2,3,4, Simon Andrews¹, Olivia Casanueva^{1 1}Babraham 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 Pagani^2,4, Silvia Di Angelantonio^2,4, Alessandro Rosa^{1,2 1}Department 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 Dourlen^1,2, Clemence Levet¹, Alexandre Mejat¹, Alexis Gambis³, Bertrand Mollereau^{1 1}Laboratory 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. Sakmar^{1 1}Laboratory 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 Collymore^1,2, Skye Rasmussen², Ravi J. Tolwani^{2 1}Tri-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.