University of Texas Southwestern Medical Center 42 articles published in JoVE Biochemistry An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation Shreenidhi Rajkumar1, Danielle Dixon1, Andrew M. Lipchik2, Joshua J. Gruber1 1Departments of Internal Medicine and the Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 2Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity. Neuroscience A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice Robert J. Pendry1,2, Lilyana D. Quigley1,2, Lenora J. Volk1,3,4, Brad E. Pfeiffer1,3 1Department of Neuroscience, UT Southwestern Medical Center, 2Neuroscience Graduate Program, UT Southwestern Medical Center, 3O’Donnell Brain Institute, UT Southwestern Medical Center, 4Department of Psychiatry, UT Southwestern Medical Center Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond. Neuroscience Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice Simone Degan1, Yu Feng1, Ulrike Hoffmann2, Dennis A. Turner1,3,4,5 1Department of Neurosurgery, Duke University Medical Center, 2Department of Anesthesiology and Pain Management, University Texas Southwestern Medical School, 3Department of Neurobiology, Duke University Medical Center, 4Department of Biomedical Engineering, Duke University, 5Research and Surgery Services, Durham VA Medical Center We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations. Neuroscience Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation Ran Li1, Weina Duan1, Dong Zhang2, Ulrike Hoffmann3, Junjie Yao2, Wei Yang1, Huaxin Sheng1 1Multidisciplinary Brain Protection Program, Department of Anesthesiology, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3Department of Anesthesiology and Pain Management, UT Southwestern University Medical Center This protocol demonstrates a unique mouse model of asphyxia cardiac arrest that does not require chest compression for resuscitation. This model is useful for monitoring and imaging the dynamics of brain physiology during cardiac arrest and resuscitation. Cancer Research Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging Serena L. Cornelius1,2, Megan M. Colonnetta2,3,4,5, Katherine E. Lake1,2, Clayton A. Smith1,2, Yu-An Zhang1,2, Evanthia T. Roussos-Torres6, Sangeetha M. Reddy1,2, Elizabeth H. Chen2,3,4,5, Isaac S. Chan1,2,3,4 1Department of Internal Medicine, Division of Hematology and Oncology, University of Texas Southwestern Medical Center, 2Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 3Department of Molecular Biology, University of Texas Southwestern Medical Center, 4Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, 5Department of Cell Biology, University of Texas Southwestern Medical Center, 6Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids. Medicine Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies Hannah E. Korah1, Kevin Cheng1, Stephanie M. Washington2, Matthew E. Flowers1, Harrison J. Stratton1, Amol Patwardhan5, Mohab M. Ibrahim*1,2,3,4, Laurent F. Martin*1,2 1Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, 2Department of Anesthesiology, College of Medicine, The University of Arizona, Tucson, 3Neuroscience Graduate Interdisciplinary Program, College of Medicine, The University of Arizona, Tucson, 4Comprehensive Pain and Addiction Center, The University of Arizona, Tucson, 5Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical Center Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility. Medicine Reduced Procedure Time and Variability with Active Esophageal Cooling During Radiofrequency Ablation for Atrial Fibrillation Christopher Joseph1, Julie Cooper2, Robert W. Turer3, Samuel A. McDonald3, Erik Benjamin Kulstad1, James Daniels4 1Department of Emergency Medicine, University of Texas Southwestern Medical Center, 2Department of Internal Medicine, University of Texas Southwestern Medical Center, 3Department of Emergency Medicine and Clinical Informatics Center, University of Texas Southwestern Medical Center, 4Department of Electrophysiology, University of Texas Southwestern Medical Center This study utilized advanced informatics techniques to compare procedure duration in patients undergoing radiofrequency atrial ablation treated with active esophageal cooling to those treated with traditional luminal esophageal temperature monitoring. Contextual inquiry, workflow analysis, and data mapping were utilized. The findings demonstrated reduced procedure time and variability with active cooling. Editorial Methods For Studying Osteoenergetics And Metabolism Elizabeth Rendina-Ruedy1, Courtney M. Karner2 1Department of Medicine, Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Internal Medicine, Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center Medicine Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts Leyao Shen1, Courtney M. Karner1,2 1Department of Internal Medicine, University of Texas Southwestern Medical Center, 2Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones. Biology Monitoring Gut Acidification in the Adult Drosophila Intestine Farhan Abu1, Benjamin Ohlstein1 1Children's Research Institute, University of Texas Southwestern Medical Center Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species. Neuroscience Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization Johnson Bob-Manuel1, Laurent Gautron1 1Center for Hypothalamic Research and Department of Internal Medicine, UTSouthwestern Medical Center at Dallas Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons. Genetics Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies Belle M. Sharon1, Neha V. Hulyalkar1, Vivian H. Nguyen1, Philippe E. Zimmern2, Kelli L. Palmer1, Nicole J. De Nisco1 1Department of Biological Sciences, University of Texas at Dallas, 2Department of Urology, University of Texas Southwestern Medical Center This protocol details a comprehensive approach for the culturing, sequencing, and de novo hybrid genome assembly of urinary bacteria. It provides a reproducible procedure for the generation of complete, circular genome sequences useful in studying both chromosomal and extrachromosomal genetic elements contributing to urinary colonization, pathogenesis, and antimicrobial resistance dissemination. Cancer Research Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates Elizabeth R. M. Zunica1,2,3, Christopher L. Axelrod1, L. Anne Gilmore2,4, John P. Kirwan1,3 1Integrated Physiology and Molecular Medicine Laboratory, Pennington Biomedical Research Center, 2Clinical Oncology and Metabolism, Pennington Biomedical Research Center, 3Department of Nutrition, Case Western Reserve University, 4Department of Clinical Nutrition, University of Texas Southwestern Medical Center We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response. Biochemistry Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography Sean Rogers1, W. Mike Henne1 1Department of Cell Biology, University of Texas Southwestern Medical Center Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids. Biology Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability Rupam Ghosh*1, Jaka Kragelj*1, Yiling Xiao*1, Kendra K. Frederick1,2 1Department of Biophysics, University of Texas Southwestern Medical Center, 2Center for Alzheimer's and Neurodegenerative Disease and Center for Systems Biology, University of Texas Southwestern Medical Center Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment. Genetics Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification Xianju Bi1,2, Xuechun Zhang1, Xiaohua Shen1 1Tsinghua-Peking Center for Life Sciences, School of Medicine and School of Life Sciences, Tsinghua University, 2Department of Molecular Biology, University of Texas Southwestern Medical Center Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells. Medicine Cooling or Warming the Esophagus to Reduce Esophageal Injury During Left Atrial Ablation in the Treatment of Atrial Fibrillation Jason Zagrodzky1, Mark M. Gallagher2, Lisa W. M. Leung2, Tiffany Sharkoski3, Pasquale Santangeli4, Cory Tschabrunn4, Jose M. Guerra5, Bieito Campos5, John MacGregor6, Jamal Hayat2, Brad Clark7, Alex Mazur8, Marcel Feher9, Martin Arnold9, Mark Metzl10, Jose Nazari10, Erik Kulstad11 1 The goal of this protocol is to describe the use of esophageal temperature modulation to counteract esophageal thermal injury from left atrial ablation for the treatment of atrial fibrillation. Biology Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization Michael L. Neugent*1, Jashkaran Gadhvi*1, Kelli L. Palmer1, Philippe E. Zimmern2, Nicole J. De Nisco1 1Department of Biological Sciences, University of Texas at Dallas, 2Department of Urology, University of Texas Southwestern Medical Center This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy. Neuroscience Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice Hisayuki Osanai1, Takashi Kitamura1,2, Jun Yamamoto1 1Department of Psychiatry, University of Texas Southwestern Medical Center, 2Department of Neuroscience, University of Texas Southwestern Medical Center This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes. Biology Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate Michael Kalwat1, Melanie H. Cobb1 1Department of Pharmacology, University of Texas Southwestern Medical Center This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes. Immunology and Infection Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors Emily A. Rex1, Dahee Seo1, Don B. Gammon1 1Department of Microbiology, University of Texas Southwestern Medical Center Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios. Biochemistry Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers Sarah Hanna-Addams1, Zhigao Wang1 1Department of Molecular Biology, UT Southwestern Medical Center Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process. Medicine Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management Melissa I. Naiman1,2, Maria Gray2, Joseph Haymore3, Ahmed F. Hegazy4, Andrej Markota5, Neeraj Badjatia6, Erik B. Kulstad2,7 1Center for Advanced Design, Research, and Exploration, University of Illinois at Chicago, 2Attune Medical, 3University of Maryland School of Nursing, 4University of Western Ontario, 5University Medical Centre Maribor, 6University of Maryland, 7Department of Emergency Medicine, University of Texas, Southwestern Medical Center This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management. Medicine Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection Hao Chen1, Robert Klein1, Stacy Arnold1, Yiying Wang2, Setsuko Chambers3,4, Wenxin Zheng5,6 1Department of Pathology, University of Arizona College of Medicine, 2 We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens. Biochemistry Assaying Protein Kinase Activity with Radiolabeled ATP Aroon S. Karra1, Steve Stippec1, Melanie H. Cobb1 1Department of Pharmacology, University of Texas Southwestern Medical Center Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([γ-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation. Developmental Biology Using Primary Neurosphere Cultures to Study Primary Cilia Issei S. Shimada1, Hemant Badgandi1, Bandarigoda N. Somatilaka1, Saikat Mukhopadhyay1 1Department of Cell Biology, University of Texas Southwestern Medical Center The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures. Developmental Biology Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts Antonio Fernandez-Perez1, Nikhil V. Munshi1 1Department of Internal Medicine- Cardiology, UT Southwestern Medical Center This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5. Immunology and Infection The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies S. M. Nashir Udden1, Sumyya Waliullah1, Melanie Harris1, Hasan Zaki1 1Department of Pathology, UT Southwestern Medical Center The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine. Biochemistry Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells Zheng Ma*1, Victor Fung*1, Iván D'Orso1 1Department of Microbiology, The University of Texas Southwestern Medical Center We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function. Biology A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing Michael Allen Stiffler1, Subu K Subramanian1, Victor H Salinas1, Rama Ranganathan1 1Green Center for Systems Biology, University of Texas Southwestern Medical Center We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 β-lactamase selected at a clinically relevant dosage of ampicillin are provided. Neuroscience Whole-cell Patch-clamp Recordings in Brain Slices Amir Segev1, Francisco Garcia-Oscos1, Saïd Kourrich1 1Department of Psychiatry, University of Texas Southwestern Medical Center This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits. Engineering Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents Christopher M. Walker1, Matthew Merritt2, Jian-Xiong Wang2, James A. Bankson1 1Imaging Physics, University of Texas M.D. Anderson Cancer Center, 2Advanced Imaging Research Center, University of Texas Southwestern Medical Center A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents. Bioengineering Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry Jennifer L. Furman*1, Brandon B. Holmes*1, Marc I. Diamond1 1Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples. Biology Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule Jeffrey N. Schellinger1, Aylin R. Rodan1 1Department of Internal Medicine, University of Texas Southwestern Medical Center This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described. Medicine Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice Katherine Poinsatte1, Uma Maheswari Selvaraj1, Sterling B. Ortega1, Erik J. Plautz1, Xiangmei Kong1, Jeffrey M. Gidday2, Ann M. Stowe1 1Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, 2Department of Neurological Surgery, Washington University School of Medicine This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection. Medicine Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling Jerome D. Robin1, Woody E. Wright1, Yaqun Zou2, Stacy A. Cossette3, Michael W. Lawlor3, Emanuela Gussoni4 1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4 This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications. Medicine MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges Hanzhang Lu1, Peiying Liu1, Uma Yezhuvath1, Yamei Cheng1, Olga Marshall2, Yulin Ge2 1Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 2Center for Biomedical Imaging, Department of Radiology, New York University School of Medicine Non-invasive imaging of the brain vasculature’s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI). Neuroscience Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb Wayne I. Doyle1, Gary F. Hammen2, Julian P. Meeks1 1Department of Neuroscience, UT Southwestern Medical Center, 2Department of Anatomy and Neurobiology, Washington University in St. Louis The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones. Biology Rapid Analysis and Exploration of Fluorescence Microscopy Images Benjamin Pavie*1, Satwik Rajaram*1, Austin Ouyang2, Jason M. Altschuler1,3, Robert J. Steininger III1, Lani F. Wu1, Steven J. Altschuler1 1Green Center for Systems Biology, UT Southwestern Medical Center, 2Advanced Imaging Research Center, UT Southwestern Medical Center, 3Princeton University Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform. Medicine Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI Heling Zhou1, Dawen Zhao1 1Radiology, University of Texas Southwestern Medical Center A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI. Biology Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies Sungsoo Lee*1, Hui Zheng*1, Liang Shi1, Qiu-Xing Jiang1 1Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins. Biology Recombineering Homologous Recombination Constructs in Drosophila Arnaldo Carreira-Rosario1, Shane Scoggin1, Nevine A. Shalaby1, Nathan David Williams1, P. Robin Hiesinger2,3, Michael Buszczak1 1Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 2Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 3Green Center for Systems Biology, University of Texas Southwestern Medical Center at Dallas Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.
