Baylor College of Medicine View Institution's Website 83 articles published in JoVE Immunology and Infection Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators J. Thomas Gebert1, Francesca J. Scribano1, Kristen A. Engevik1, Joseph M. Hyser1 1Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis. Behavior Screening People on Standing Balance with Romberg Testing and Walking Balance with Tandem Walking Helen S. Cohen1 1Bobby R Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine This article describes procedures for screening people for standing and walking balance impairments using two normed, rapid, low-tech balance tests. Medicine Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography Anish Attarde1, Thomas S. Riad1, Zhao Zhang1, Manisha Ahir1, Yingbin Fu1 1Cullen Eye Institute, Baylor College of Medicine Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models. Biology A Mouse Model for Corneal Neovascularization by Alkali Burn Remya Ammassam Veettil1, Wei Li1, Stephen C. Pflugfelder1, Douglas D. Koch1 1Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization. Biology Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome Fan Zhang*1, Dana Blackburn*1, Ciara N. Hosea1,2, Adrien Assié1, Buck S. Samuel1,2 1Alkek Center for Metagenomics and Microbiome Research and Department of Molecular Virology and Microbiology, Baylor College of Medicine, 2Development, Disease Models and Therapeutics Program, Graduate School of Biomedical Sciences, Baylor College of Medicine Caenorhabditis elegans is a powerful model to examine the molecular determinants driving host-microbiome interactions. We present a high throughput pipeline profiling the single animal levels of gut microbiome colonization together with key aspects of the C. elegans physiology. Cancer Research In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection Wen Bu1,2, Yi Li1,3 1Lester & Sue Smith Breast Center, Baylor College of Medicine, 2Department of Medicine, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030 The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells. Biology Establishing 3D Endometrial Organoids from the Mouse Uterus Suni Tang1,2, Sydney E. Parks1,2, Zian Liao1,2, Dominique I. Cope1,2, Sarah E. Blutt3, Diana Monsivais1,2 1Department of Pathology & Immunology, Baylor College of Medicine, 2Center for Drug Discovery, Baylor College of Medicine, 3Departments of Molecular Virology and Microbiology and Medicine, Baylor College of Medicine This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses. Biology Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans Marzia Savini*1,2, Yi-Tang Lee2,3,4, Meng C. Wang2,4,5, Yue Zhou*2 1Graduate Program in Developmental Biology, Baylor College of Medicine, 2Huffington Center on Aging, Baylor College of Medicine, 3Integrative Program of Molecular and Biochemical Sciences, Baylor College of Medicine, 4Department of Molecular and Human Genetics, Baylor College of Medicine, 5Howard Hughes Medical Institute, Baylor College of Medicine The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues. Immunology and Infection Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages Beatriz E. Bolívar1, Lisa Bouchier-Hayes1 1Department of Pediatrics, Division of Hematology-Oncology, Department of Molecular and Cellular Biology, Texas Children’s Hospital William T. Shearer Center for Human Immunobiology, Baylor College of Medicine This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells. Medicine Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model Alexandra L. Gannon1,2, Shaji K. Chacko3, Inka C. Didelija3, Juan C. Marini3,4, Chellakkan S. Blesson1,2 1Department of Reproductive Endocrinology and Infertility, Baylor College of Medicine, 2Family Fertility Center, Texas Children’s Hospital, 3Children’s Nutrition Research Center, Baylor College of Medicine, 4Critical Care Medicine, Department of Pediatrics, Baylor College of Medicine This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem. Medicine Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research Vojtech Gabriel1, Christopher Zdyrski1, Dipak K. Sahoo2, Kimberly Dao3, Agnes Bourgois-Mochel2, Jamie Kopper2, Xi-Lei Zeng4, Mary K. Estes4, Jonathan P. Mochel1,3, Karin Allenspach2,3 1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, 33D Health Solutions Inc., 4Department of Molecular Virology and Microbiology, Baylor College of Medicine Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed. Medicine An Epithelial Abrasion Model for Studying Corneal Wound Healing Prince K. Akowuah1, Angie De La Cruz1, C. Wayne Smith2, Rolando E. Rumbaut2,3, Alan R. Burns1,2 1College of Optometry, University of Houston, 2Children’s Nutrition Research Center, Baylor College of Medicine, 3Center for Translational Research on Inflammatory Diseases (CTRID), Michael E. DeBakey Veterans Affairs Medical Center Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases. Neuroscience Free-floating Immunostaining of Mouse Brains Longlong Tu1, Nan Zhang1,2,3, Kristine M Conde1, Jonathan C Bean1, Chunmei Wang1, Yong Xu1,4 1USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 2Department of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 3Hubei Provincial Clinical Research Center for Diabetes and Metabolic Disorder, 4Department of Molecular and Cellular Biology, Baylor College of Medicine This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging. Developmental Biology Generation of Naïve Blastoderm Explants from Zebrafish Embryos Alyssa Alaniz Emig1, Margot L. K. Williams1 1Center for Precision Environmental Health and Department of Molecular and Cellular Biology, Baylor College of Medicine Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively naïve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation. Biology Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy Ayse Sena Mutlu1, Tao Chen1, Dinghuan Deng1, Meng C. Wang1,2,3 1Huffington Center on Aging, Baylor College of Medicine, 2Department of Molecular and Human Genetics, Baylor College of Medicine, 3Howard Hughes Medical Institute, Baylor College of Medicine Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans. Neuroscience Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing Brandon T. Pekarek1, Patrick J. Hunt1,2, Benjamin D. W. Belfort1,2, Gary Liu2, Benjamin R. Arenkiel1,3 1Department of Genetics and Genomics, Baylor College of Medicine, 2Medical Scientist Training Program, Baylor College of Medicine, 3Department of Neuroscience, Baylor College of Medicine Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines. Biology Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples Justin A. Courson1, Paul T. Landry1, Thao Do1, Eric Spehlmann2, Pascal J. Lafontant2, Nimesh Patel1, Rolando E. Rumbaut3,4, Alan R. Burns1,4 1College of Optometry, University of Houston, 2Department of Biology, DePauw University, 3Center for Translational Research on Inflammatory Diseases (CTRID), Michael E. DeBakey Veterans Affairs Medical Center, 4Children’s Nutrition Center, Baylor College of Medicine This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process. Medicine Assessing Whole-Body Lipid-Handling Capacity in Mice Mingyang Huang1, Noah Mathew1, Yi Zhu1 1Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine This paper provides three easy and accessible assays for assessing lipid metabolism in mice. Immunology and Infection Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System Daniel E. Morales-Mantilla1,2, Xinyan Huang3,7, Philip Erice1,3, Paul Porter4, Yun Zhang1,5, Mary Figueroa6, Joya Chandra6, Katherine Y. King2, Farrah Kheradmand4,8, Antony Rodríguez3,8 1Program in Immunology, Baylor College of Medicine, 2Department of Pediatrics, Section of Infectious Diseases, Baylor College of Medicine, 3Department of Medicine - Immunology Allergy and Rheumatology, Baylor College of Medicine, 4Department of Medicine, Pulmonary, Critical Care, Sleep Medicine, Baylor College of Medicine, 5Department of Pathology and Immunology, Baylor College of Medicine, 6Department of Pediatrics, Research and Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, 7Division of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Sun Yat-sen University, 8Center for Translational Research on Inflammatory Diseases (CTRID), Michael E. DeBakey VA Medical Center This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis. Developmental Biology Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos Rachel A. Keuls1, Ronald Parchem1 1Development, Disease Models & Therapeutics Graduate Program, Department of Molecular and Cellular Biology, Department of Neuroscience, Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo. Biochemistry Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH Ragini M. Mistry1,2, Pankaj K. Singh1,3, Maureen G. Mancini1,2, Fabio Stossi1,2, Michael A. Mancini1,2,3,4 1GCC Center for Advanced Microscopy and Image Informatics, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, 3Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, 4Department of Pharmacology and Chemical Biology, Baylor College of Medicine Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs. Developmental Biology Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice Laura Ortinau1,2, Kevin Lei1, Youngjae Jeong1, Dongsu Park1,2,3 1Department of Molecular & Human Genetics, Baylor College of Medicine, 2Center for Skeletal Biology, Baylor College of Medicine, 3Department of Pathology & Immunology, Baylor College of Medicine This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy. Cancer Research Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer Xiangdong Lv*1,2,3, Lacey E. Dobrolecki*1,2,3, Yao Ding1,2,3, Jeffrey M. Rosen1,2,3, Michael T. Lewis1,2,3, Xi Chen1,2,3 1Department of Molecular and Cellular Biology, Baylor College of Medicine, 2Lester and Sue Smith Breast Center, Baylor College of Medicine, 3Dan L. Duncan Cancer Center, Baylor College of Medicine Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response. Developmental Biology Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Natalie Biel1,2, Lauren Figard3, Anna Marie Sokac1,2,3 1Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, 2Department of Cell and Molecular Biology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 3Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress. Medicine Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry Minke A.E. Rab1,2, Brigitte A. van Oirschot1, Jennifer Bos1, Celeste K. Kanne3, Vivien A. Sheehan3, Eduard J. van Beers2, Richard van Wijk1 1Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University, 2Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, 3Department of Pediatrics, Division of Hematology/Oncology, Baylor College of Medicine Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy. Biology A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice Daniel Villarreal1, Geetali Pradhan2, Chia-Shan Wu1,2, Clinton D Allred1, Shaodong Guo1, Yuxiang Sun1,2 1Department of Nutrition and Food Science, Texas A&M University, 2Children's Nutrition Research Center, Baylor College of Medicine This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse. 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. Immunology and Infection High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model Howard W. Davidson1, Joseph Ray Cepeda2, Nitin S. Sekhar2, Junying Han2, Ling Gao3, Tomasz Sosinowski1, Li Zhang2 1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 2Department of Medicine, Endocrinology, Diabetes & Metabolism, Baylor College of Medicine, 3Scientific Center, Shandong Provincial Hospital affiliated to Shandong University This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo. 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. Immunology and Infection Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers Alyssa C. Fasciano1, Sarah E. Blutt2, Mary K. Estes2, Joan Mecsas3 1Program in Immunology, Sackler School of Graduate Biomedical Sciences, 2Department of Molecular Virology and Microbiology, Baylor College of Medicine, 3Department of Molecular Biology and Microbiology, Tufts University This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development. Developmental Biology Determining Bile Duct Density in the Mouse Liver Joshua M. Adams1,2,3, Hamed Jafar-Nejad1,3 1Program in Developmental Biology, Baylor College of Medicine, 2Medical Scientist Training Program (MSTP), Baylor College of Medicine, 3Department of Molecular and Human Genetics, Baylor College of Medicine We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases. Medicine Remote Laboratory Management: Respiratory Virus Diagnostics Elena V. Petrova1,2, Vasanthi Avadhanula3, Sarah Michel1, Karen E. Gincoo3, Pedro A. Piedra3, Sharmila Anandasabapathy1,2 1Baylor Global Health, Baylor College of Medicine, 2Department of Medicine, Baylor College of Medicine, 3Department of Molecular Virology and Microbiology, Baylor College of Medicine A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented. Genetics Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation Edward G Jones1, Andrew P Landstrom2 1Department of Pediatrics, Baylor College of Medicine, 2Department of Pediatrics, Division of Cardiology, Duke University School of Medicine Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant "hotspots" within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise). Genetics Culturing and Manipulation of O9-1 Neural Crest Cells Bao H. Nguyen1, Mamoru Ishii2, Robert E. Maxson2, Jun Wang1 1Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Biochemistry and Molecular Biology, University of Southern California O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing. Cancer Research Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors Jared M. Newton*1,2, Aurelie Hanoteau*1, Andrew G. Sikora1 1Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, 2Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment. Developmental Biology Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro Marissa A. Scavuzzo1, Jessica Teaw2,3,4, Diane Yang2,3,4, Malgorzata Borowiak1,2,3,4,5 1Program in Developmental Biology, Baylor College of Medicine, 2Center for Cell and Gene Therapy, Texas Children's Hospital, and Houston Methodist Hospital, Baylor College of Medicine, 3Molecular and Cellular Biology Department, Baylor College of Medicine, 4Stem Cell and Regenerative Medicine Center, Baylor College of Medicine, 5McNair Medical Institute, Baylor College of Medicine Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme. Neuroscience Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats Li Lu1, Briana Popeney1, J. David Dickman1, Dora E. Angelaki1 1Department of Neuroscience, Baylor College of Medicine We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks. 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. Neuroscience An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice Gary Liu*1,2, Jay M. Patel*2,3, Burak Tepe1, Cynthia K. McClard2,4, Jessica Swanson4, Kathleen B. Quast4, Benjamin R. Arenkiel1,3,4,5 1Program in Developmental Biology, Baylor College of Medicine, 2Medical Scientist Training Program, Baylor College of Medicine, 3Department of Neuroscience, Baylor College of Medicine, 4Department of Molecular and Human Genetics, Baylor College of Medicine, 5 Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain. Biochemistry Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation Chloé I Charendoff1, Lisa Bouchier-Hayes2 1Department of Pediatrics, Division of Hematology-Oncology, Baylor College of Medicine, 2Department of Pediatrics, Division of Hematology-Oncology and Department of Molecular and Cellular Biology, Baylor College of Medicine This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation. Biology Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney Lauren E. Woodard1,2,3, Richard C. Welch2, Felisha M. Williams2, Wentian Luo2, Jizhong Cheng3, Matthew H. Wilson1,2,3 1Department of Veterans Affairs, Tennessee Valley Healthcare System, 2Departments of Medicine and Pharmacology, Vanderbilt University Medical Center, 3Department of Medicine, Baylor University College of Medicine This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney. Developmental Biology Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands Ashutosh Pandey1, Hamed Jafar-Nejad1,2 1Department of Molecular and Human Genetics, Baylor College of Medicine, 2Program in Developmental Biology, Baylor College of Medicine Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands. Developmental Biology A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse Mary C. Peavey1,4, Corey L. Reynolds2, Maria M. Szwarc2, William E. Gibbons1, Cecilia T. Valdes1, Francesco J. DeMayo*3, John P. Lydon*4 1Devision of Repreoductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Baylor College of Medicine, 2Mouse Phenotyping Core, Baylor College of Medicine, 3Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, 4Department of Molecular and Cellular Biology, Baylor College of Medicine Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse. Medicine Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition Sidharth Tyagi1, Donald Beqollari1, Chang Seok Lee2, Lori A. Walker1, Roger A. Bannister1 1Department of Medicine-Cardiology Division, University of Colorado School of Medicine, 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology. Medicine Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery Alejandro Granillo1, Celia A Pena1, Thuy Pham1, Lavannya M Pandit2, George E. Taffet1 1Huffington Center on Aging, Baylor College of Medicine, 2Pulmonary/Critical Care/Sleep Medicine, Michael E. DeBakey Veterans Affairs Medical Center The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function. Developmental Biology Spatial and Temporal Analysis of Active ERK in the C. elegans Germline Amanda L. Gervaise1, Swathi Arur1,2 1Program in Developmental Biology, Baylor College of Medicine, 2Department of Genetics, UT MD Anderson Cancer Center We present an immunofluorescence imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available. Neuroscience Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina Hung-Ya Tu1, Chih-Chun Hsu1,2, Yu-Jiun Chen1, Ching-Kang Chen1,2,3 1Department of Ophthalmology, Baylor College of Medicine, 2Department of Neuroscience, Baylor College of Medicine, 3Department of Biochemistry and Molecular Biology, Baylor College of Medicine This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation. Medicine Exergaming in Older People Living with HIV Improves Balance, Mobility and Ameliorates Some Aspects of Frailty Suhitha Veeravelli1, Bijan Najafi3, Ivan Marin3, Fernando Blumenkron2, Shannon Smith2, Stephen A. Klotz2 1Department of Surgery, Interdisciplinary Consortium on Advanced Motion Performance (iCAMP), College of Medicine, University of Arizona, 2Department of Medicine, Division of Infectious Disease, College of Medicine, University of Arizona, 3Interdisciplinary Consortium on Advanced Motion Performance (iCAMP), Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine Persons infected with HIV are often frail, depressed and live a sedentary lifestyle for which conventional exercise is too taxing. Here, we present an exercise protocol that ameliorates aspects of frailty in HIV-infected persons. An exergame integrating cognitive control was developed using biosensors that measured balance, weight-shifting and obstacle crossing. Cancer Research Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis Cuijuan Yu1,2, Hai Wang1,2, Aaron Muscarella1,2, Amit Goldstein1,2, Huan-Chang Zeng3, Yangjin Bae4, Brendan H. I. Lee4, Xiang H.-F. Zhang1,2,5,6 1Lester and Sue Smith Breast Center, Baylor College of Medicine, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, 3Graduate Program in Developmental Biology, Baylor College of Medicine, 4Department of Molecular and Human Genetics, Baylor College of Medicine, 5McNair Medical Institute, Baylor College of Medicine, 6Dan L. Duncan Cancer Center, Baylor College of Medicine This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types. Immunology and Infection Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice Thomas Lee1, Ivan Shevchenko1, Maran L. Sprouse1, Maria Bettini1, Matthew L. Bettini1 1Department of Pediatrics, Baylor College of Medicine We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice. Developmental Biology Isolation of Murine Embryonic Hemogenic Endothelial Cells Jennifer S. Fang*1, Emily C. Gritz*2, Kathrina L. Marcelo3, Karen K. Hirschi1 1Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine, 2Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Yale University School of Medicine, 3Department of Molecular and Cellular Biology, Baylor College of Medicine Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues. Medicine The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma Michael Li1,2, Ravi R. Pathak5, Esther Lopez-Rivera3, Scott L. Friedman1, Julio A. Aguirre-Ghiso4, Andrew G. Sikora5 1Department of Medicine, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, 2Department of Otolaryngology, Icahn School of Medicine at Mount Sinai, 3Division of Nephrology, Columbia University College of Physicians and Surgeons, 4Departments of Medicine, Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, 5Bobby R. Alford Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay. Medicine Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy Maxime M. Mahe1, Nambirajan Sundaram1, Carey L. Watson1, Noah F. Shroyer2, Michael A. Helmrath1 1 We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids. 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. Behavior Topographical Estimation of Visual Population Receptive Fields by fMRI Sangkyun Lee1, Amalia Papanikolaou2, Georgios A. Keliris2,3, Stelios M. Smirnakis1 1Department of Neuroscience and Neurology, Baylor College of Medicine, 2Max Planck Institute for Biological Cybernetics, 3Bernstein Center for Computational Neuroscience It is important to obtain unbiased estimates of visual population receptive fields (pRFs) by functional magnetic resonance imaging. We use mild regularization constraints to estimate pRF topography without a-priori assumptions about pRF shape, allowing us to choose specific pRF models post-hoc. This is particularly advantageous in subjects with visual-pathway lesions. Neuroscience Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies Claudia Kathe1, Thomas H. Hutson1, Qin Chen2, Harold D. Shine2, Stephen B. McMahon1, Lawrence D. F. Moon1 1 The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (“pyramidotomy”) and postoperative assessments are described in this protocol. Neuroscience Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction Ji-Yoen Kim*1, Stacy D. Grunke*1, Yona Levites2, Todd E. Golde2, Joanna L. Jankowsky1,3 1Department of Neuroscience, Baylor College of Medicine, 2Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, University of Florida, 3Department of Neurology and Department of Neurosurgery, Baylor College of Medicine Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes. Medicine Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy Zulfi Haneef1,2, Agatha Lenartowicz3, Hsiang J. Yeh4, Jerome Engel Jr.4, John M. Stern4 1Department of Neurology, Baylor College of Medicine, 2Neurology Care Line, Michael E. DeBakey VA Medical Center, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, 4Department of Neurology, University of California, Los Angeles The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI). 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. Medicine Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes Candace L. Minchew1,2, Vladimir V. Didenko1,2 1Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death. Neuroscience Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography Sarah H. Shahmoradian1, Mauricio R. Galiano2, Chengbiao Wu3, Shurui Chen4, Matthew N. Rasband2, William C. Mobley3, Wah Chiu4 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Neuroscience, Baylor College of Medicine, 3Department of Neuroscience, University of California at San Diego, 4National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale. Immunology and Infection piggyBac Transposon System Modification of Primary Human T Cells Sunandan Saha1,2, Yozo Nakazawa3, Leslie E. Huye4,5, Joseph E. Doherty2,6, Daniel L. Galvan2, Cliona M. Rooney4,5,7, Matthew H. Wilson1,2,4,8 1Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, 2Department of Medicine, Division of Nephrology, Baylor College of Medicine, 3Department of Immunology and Pathology, Shinshu University School of Medicine, 4Center for Cell and Gene Therapy, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine, 6Program in Cell and Molecular Biology, Baylor College of Medicine, 7Department of Molecular Virology and Microbiology, Baylor College of Medicine, 8Michael E. DeBakey VA Medical Center We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression. 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. Medicine Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer Rongying Li1,2, Kazuhiro Oka1,2,3, Vijay Yechoor1,2,3 1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression. Medicine Analytical Techniques for Assaying Nitric Oxide Bioactivity Hong Jiang1, Deepa Parthasarathy1, Ashley C. Torregrossa1, Asad Mian2, Nathan S. Bryan1 1Texas Therapeutics Institute, University of Texas Health Science Center at Houston, 2Deptartment of Pediatrics, Baylor College of Medicine The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease. Immunology and Infection Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus Patrick J. Hanley1, Sharon Lam1,2, Elizabeth J. Shpall3, Catherine M. Bollard1,2,4,5 1Center for Cell and Gene Therapy, Baylor College of Medicine, 2Pathology and Immunology, Baylor College of Medicine, 3Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, 4Medicine, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly naîve T cells. Bioengineering Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model Kevin J. Zwezdaryk*1, Jessica A. Warner*1,2, Heather L. Machado3, Cindy A. Morris1, Kerstin Höner zu Bentrup1 1Department of Microbiology and Immunology, Tulane University Medical School, 2Physician/Scientist Program, Tulane University Medical School, 3Department of Molecular and Cellular Biology, Baylor College of Medicine Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line. Bioengineering In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage Candace L. Minchew1,2, Vladimir V. Didenko1,2,3 1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends. Medicine Production and Detection of Reactive Oxygen Species (ROS) in Cancers Danli Wu1, Patricia Yotnda1 1Center for Cell and Gene Therapy, Baylor College of Medicine Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells. Immunology and Infection Following Cell-fate in E. coli After Infection by Phage Lambda Lanying Zeng1, Ido Golding1,2,3 1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results. Biology Induction and Testing of Hypoxia in Cell Culture Danli Wu1, Patricia Yotnda1 1Center for Cell and Gene Therapy, Baylor College of Medicine Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures. Medicine Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice Wesley S. Bond1,2, Lalita Wadhwa2,3, Laszlo Perlaky2,3, Rebecca L. Penland4, Mary Y. Hurwitz2,3, Richard L. Hurwitz*2,3,5,6, Patricia Chèvez-Barrios*4,5,7 1Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor College of Medicine, 2Texas Children's Cancer Center, Baylor College of Medicine, 3Department of Pediatrics, Baylor College of Medicine, 4Department of Pathology, The Methodist Hospital Research Institute, 5Department of Ophthalmology, Retinoblastoma Center of Houston, 6Baylor College of Medicine, Center for Cell and Gene Therapy, 7Center for Cell and Gene Therapy, Baylor College of Medicine A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres. 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. Immunology and Infection Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant Ulrike Gerdemann1, Juan F. Vera1, Cliona M. Rooney1, Ann M. Leen1 1Center for Cell and Gene Therapy, Baylor College of Medicine A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers. Neuroscience Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation Onkar S. Dhande1,2, Michael C. Crair1 1Department of Neurobiology, Yale University, 2Program in Developmental Biology, Baylor College of Medicine We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies. Biology Imaging Cell Shape Change in Living Drosophila Embryos Lauren Figard1, Anna Marie Sokac1,2 1Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), 2Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine (BCM) Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization. Biology Detection of Functional Matrix Metalloproteinases by Zymography Xueyou Hu1, Christine Beeton1 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids. Biology Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry Shyny Koshy1, Parema Alizadeh1, Lubov T. Timchenko1, Christine Beeton1 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry. Medicine Transthoracic Echocardiography in Mice Jonathan L. Respress1, Xander H.T. Wehrens1,2 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM) Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance. Biology Ambulatory ECG Recording in Mice Mark D. McCauley1,2, Xander H.T Wehrens1,2 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM) Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice. Medicine Programmed Electrical Stimulation in Mice Na Li1, Xander H.T Wehrens1,2 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM) Programmed electrical stimulation provides the ability to determine conduction properties of the heart, and the possibility to induce and terminate cardiac arrhythmias using various pacing protocols. Using a transvenous catheter, intracardiac electrogram recordings can be obtained in mice following programmed electrical stimulation protocols to identify arrhythmogenic substrates. Biology Transverse Aortic Constriction in Mice Angela C. deAlmeida1, Ralph J. van Oort1, Xander H.T. Wehrens1,2 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM) Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice. Biology A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice Sumanth Polikepahad1, Wade T. Barranco1, Paul Porter1, Bruce Anderson2, Farrah Kheradmand1,3, David B. Corry1,3 1Department of Medicine, Baylor College of Medicine (BCM), 2Millenium Premier Group, 3Department of Immunology, Baylor College of Medicine (BCM) Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.
