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Cincinnati Children's Hospital Medical Center

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39 articles published in JoVE

Biology

An Integrated Approach for Microprotein Identification and Sequence Analysis Omar Brito-Estrada*¹, Keira R. Hassel*¹, Catherine A. Makarewich^1,2 ¹ The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.
Biology

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System Md Nasimuzzaman^1,2, Sophia Villaveces¹, Johannes C. M. van der Loo^1,2,3, Sivani Alla¹ ¹Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.
Biochemistry

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes Daniel Barrera¹, Sierra Upton², Megan Rauch³, Tara Notarianni⁴, Ki Suk Eum⁵, Megan Liberty⁶, Sarmila Venkoba Sah⁷, Robert Liu⁸, Sean Newcomer⁹, Linda E. May¹⁰, Emre Agbas¹¹, Jessica Sage¹², Edina Kosa⁷, Abdulbaki Agbas^7,13 ¹AdventHelath, ²University of Illinois Chicago School of Public Health, ³Lincoln Memorial University, ⁴Cincinnati Children’s Hospital Medical Center, ⁷Kansas City University, ⁸Roblex Rex Veterans Affairs Medical Center, ⁹California State University San Marcos, ¹⁰East Carolina University, ¹¹Massachusetts Institute of Technology, ¹²Boehringer Ingelheim Norway KS, ¹³Heartland Center for Mitochondrial Medicine Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.
Developmental Biology

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish M. Fethullah Simsek^1,3, Ertugrul M. Özbudak^1,2,3 ¹ Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.
Behavior

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants Sandra P. Zoubovsky^1,2,3, Akil Wilder⁴, Louis Muglia^1,2,3 ¹Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, ³Department of Pediatrics, University of Cincinnati College of Medicine, ⁴ The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.
Medicine

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts Demetria M. Fischesser^1,2, Evan C. Meyer³, Michelle Sargent², Jeffery D. Molkentin² ¹Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, ² A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.
Developmental Biology

Transuterine Fetal Tracheal Occlusion Model in Mice Emrah Aydın^1,2, Rashika Joshi³, Marc Oria¹, Foong-Yen Lim¹, Brian Michael Varisco³, Jose Luis Peiro¹ ¹The Division of Critical Care Medicine, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.
Developmental Biology

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye Yu-Han Yeh*^1,2, Mengwen Hu*^1,2, Toshinori Nakagawa^3,4, Akihiko Sakashita^1,2, Shosei Yoshida^3,4, So Maezawa^5,6, Satoshi H. Namekawa^1,2 ¹ Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.
Developmental Biology

A Patient-Derived Xenograft Model for Venous Malformation Sandra Schrenk*¹, Jillian Goines*¹, Elisa Boscolo^1,2 ¹ We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.
Developmental Biology

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart Alyssa Klein^1,2,3, Bethel Bayrau^1,2,3, Yifei Miao^1,2,3,4,5, Mingxia Gu^1,2,3,4,5 ¹Department of Pediatrics, Division of Cardiology, Stanford School of Medicine, ²Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford School of Medicine, ³Stanford Cardiovascular Institute, Stanford School of Medicine, ⁴ We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.
Genetics

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers Vishnu Modur¹, Navneet Singh¹, Belal Muhammad¹ ¹ The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TE^deff phenomenon, present in about 20% of all cancer types.
Biology

Purification of Platelets from Mouse Blood Marilou G. Narciso¹, Md Nasimuzzaman^1,2 ¹ Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.
Neuroscience

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5 Shuiqing Chi¹, Mijing Fang¹, Kang Li¹, Li Yang², Shao-tao Tang¹ ¹Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, ² This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung's disease has preferable sensitivity and specificity rates.
Cancer Research

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence Meenu Kesarwani¹, Zachary Kincaid¹, Mohammad Azam¹ ¹ Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.
Bioengineering

Novel Process for 3D Printing Decellularized Matrices Stacey M. S. Gruber¹, Paulomi Ghosh², Karl Wilhelm Mueller², Patrick W. Whitlock^1,2,3, Chia-Ying Lin^1,3 ¹Department of Biomedical Engineering, University of Cincinnati, ² This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.
Medicine

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method Esam S.B. Salem*^1,2, Kazutoshi Murakami*², Toshimasa Takahashi², Elise Bernhard², Vishnupriya Borra², Mridula Bethi², Takahisa Nakamura^2,3,4 ¹Department of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, ² Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.
Biochemistry

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells Xiongwei Cai^1,2,3, Yi Zheng¹, Nancy A. Speck² ¹ A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.
Medicine

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study John J. Brewington¹, Erin T. Filbrandt¹, Francis J. LaRosa III¹, Jessica D. Moncivaiz¹, Alicia J. Ostmann¹, Lauren M. Strecker¹, John P. Clancy¹ ¹ Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.
Genetics

Production and Purification of Baculovirus for Gene Therapy Application Md Nasimuzzaman^1,2, Johannes C.M. van der Loo^1,2,3, Punam Malik^1,2 ¹ In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.
Biology

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing Ayelet Di Segni¹, Tzipi Braun¹, Marina BenShoshan¹, Sarit Farage Barhom¹, Efrat Glick Saar¹, Karen Cesarkas¹, James E. Squires², Nathan Keller^1,3, Yael Haberman^1,4 ¹Sheba Medical Center, ² This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.
Neuroscience

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition Michael D. Guthrie¹, Donald L. Gilbert², David A. Huddleston², Ernest V. Pedapati^2,3, Paul S. Horn², Stewart H. Mostofsky⁴, Steve W. Wu² ¹College of Medicine, University of Cincinnati, ² We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.
Developmental Biology

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish Jillian L. Ellis¹, Chunyue Yin^1,2 ¹ This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.
Medicine

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease Victoria N. Jensen*¹, Shannon H. Romer*², Sarah M. Turner³, Steven A. Crone³ ¹Neuroscience Graduate Program, University of Cincinnati, ²Department of Biological Sciences, Wright State University, ³ This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.
Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA) Daniel E. Miller¹, Zubin H. Patel^1,2,3, Xiaoming Lu^1,3, Arthur T. Lynch¹, Matthew T. Weirauch*^1,4, Leah C. Kottyan*¹ ¹Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.
Developmental Biology

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells Ashley Ulm¹, Christopher N. Mayhew², Jason Debley³, Gurjit K. Khurana Hershey⁴, Hong Ji^1,4 ¹Division of Developmental Biology, Cincinnati Children's Hospital, ³Division of Asthma Research, Cincinnati Children's Hospital This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).
Medicine

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS Touraj Shokati¹, Nicholas Bodenberger¹, Holly Gadpaille¹, Björn Schniedewind¹, Alexander A. Vinks², Wenlei Jiang³, Rita R. Alloway⁴, Uwe Christians¹ ¹iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus, ² Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.
Medicine

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice Jared Klarquist¹, Edith M. Janssen¹ ¹ The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.
Developmental Biology

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis Sarah J. Potter¹, Tony DeFalco¹ ¹ The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.
Behavior

Vision Training Methods for Sports Concussion Mitigation and Management Joseph F. Clark¹, Angelo Colosimo², James K. Ellis³, Robert Mangine³, Benjamin Bixenmann⁴, Kimberly Hasselfeld², Patricia Graman⁵, Hagar Elgendy², Gregory Myer⁶, Jon Divine² ¹Neurology and Rehabilitative Medicine, University of Cincinnati, ²Division of Sports Medicine, Department of Orthopaedic Surgery, University of Cincinnati, ³Department of Athletics, University of Cincinnati, ⁴Department of Neurosurgery, University of Cincinnati, ⁵College of Education, Criminal Justice, and Human Services, University of Cincinnati, ⁶ This paper describes a protocol to conduct, quantitatively monitor, and assess the success of vision training initiated as part of a sports medical management program including intervention for concussion prevention and performance enhancement.
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.
Biology

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae Sheldon G. Watts*¹, Justin J. Crowder*¹, Samuel Z. Coffey^1,2, Eric M. Rubenstein¹ ¹Department of Biology, Ball State University, ² This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.
Medicine

Mouse Pneumonectomy Model of Compensatory Lung Growth Sheng Liu¹, Jeffrey Cimprich¹, Brian M. Varisco¹ ¹ Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.
Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation Minghui Yue*^1,2, John Lalith Charles Richard*^1,2, Norishige Yamada^1,2, Akiyo Ogawa¹, Yuya Ogawa^1,2 ¹ We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.
Biology

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract Tony W. Chen*¹, Matthew R. Broadus*¹, Stacey S. Huppert², Ethan Lee^1,3 ¹Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, ² A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.
Biology

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila Tadahiro Goda¹, Jennifer R. Leslie¹, Fumika N. Hamada^1,2 ¹ We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here we describe the design and analysis of TPR in Drosophila.
Biology

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells Satoshi H. Namekawa^1,2 ¹ The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).
Medicine

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System Teagan J. Walter^1,2, Erin E. Sparks³, Stacey S. Huppert² ¹Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, ²Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, ³Department of Biology, Duke University A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.
Biology

Single Cell Fate Mapping in Zebrafish Vikram Kohli¹, Kira Rehn¹, Saulius Sumanas^1,2 ¹Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, ²Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.
Medicine

Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking Kan N. Hor¹, Rolf Baumann², Gianni Pedrizzetti³, Gianni Tonti³, William M. Gottliebson¹, Michael Taylor¹, D. Woodrow Benson¹, Wojciech Mazur⁴ ¹The Heart Institute, Cincinnati Children Hospital Medical Center (CCHMC), ²TomTec, Imaging Systems GmbH, ³AMID, Advanced Medical Imaging Development SRL, ⁴The Heart and Vascular Center, The Christ Hospital An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF.

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