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Utah State University

23 ARTICLES PUBLISHED IN JoVE

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Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Shenglan Li *1,2, Haipeng Xue *1,2, Bo Long 1,2,5, Li Sun 1,2,6, Tai Truong 1,2,4,7, Ying Liu 1,2,3
1Department of Neurosurgery, The University of Texas Health Science Center at Houston, 2Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 3The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 4Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston, 5Department of Anesthesiology, Shengjing Hospital, China Medical University, 6Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 7Biology Department, University of West Georgia

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

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Environment

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae
Katerine Napan 1, Derek Hess 1, Brian McNeil 1, Jason C. Quinn 1
1Mechanical and Aerospace Engineering, Utah State University

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

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Immunology and Infection

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses
Sang-Im Yun 1, Byung-Hak Song 1, Jin-Kyoung Kim 1, Young-Min Lee 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

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Behavior

Use of Galvanic Skin Responses, Salivary Biomarkers, and Self-reports to Assess Undergraduate Student Performance During a Laboratory Exam Activity
Idalis Villanueva 1, Maria Valladares 1, Wade Goodridge 1
1Department of Engineering Education, Utah State University

Research on undergraduate students' academic achievement emotions have primarily relied on self-reports in laboratory settings. Studies rarely include bio-physiological measures to these self-reports. This protocol will describe a methodology that integrates self-reports with and bio-physiological measures to assess student response and performance during a laboratory examination activity.

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Behavior

Utilizing Electroencephalography Measurements for Comparison of Task-Specific Neural Efficiencies: Spatial Intelligence Tasks
Benjamin J. Call 1, Wade Goodridge 1, Idalis Villanueva 1, Nicholas Wan 2, Kerry Jordan 2
1Department of Engineering Education, Utah State University, 2Department of Psychology, Utah State University

This manuscript describes an approach to measure neural activity of humans while solving spatially focused engineering problems. The electroencephalogram methodology helps interpret beta brain wave measurements in terms of neural efficiency, with the aim of ultimately enabling comparisons of task performance both between problem types and between participants.

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Behavior

Measuring Delay Discounting in Humans Using an Adjusting Amount Task
Charles C.J. Frye 1, Ann Galizio 1, Jonathan E. Friedel 1, W. Brady DeHart 1, Amy L. Odum 1
1Department of Psychology, Utah State University

Delay discounting refers to a decline in the value of a reward when it is delayed relative to when it is immediately available. We outline a computer-based delay discounting task that is easy to implement and allows for the quantification of the degree of delay discounting in human participants.

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Biochemistry

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform
Cynthia Hanson *1, Nathan D. Israelsen *1, Michael Sieverts 1, Elizabeth Vargis 1
1Biological Engineering Department, Utah State University

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

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Bioengineering

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex
Rong Li *1, Jinxin Miao *1,2, Zhiqiang Fan 1, SeokHwan Song 3, Il-keun Kong 3,4, Yaohe Wang 2,5, Zhongde Wang 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2National Centre for International Research in Cell and Gene Therapy, Sino-British Research Centre, School of Basic Medical Sciences, Zhengzhou University, 3Department of Animal Science Division of Applied Life Science (BK21 Plus), Gyeongsang National University, 4Institute of Agriculture and Life Science, Gyeongsang National University, 5Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

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Bioengineering

Visual Detection of Multiple Nucleic Acids in a Capillary Array
Jianwei Chen *1,2,3, Ning Shao *1,2,3, Jiaying Hu *4, Rong Li 4, Yuanshou Zhu 1,2,3, Dabing Zhang 4,5, Shujuan Guo 1,2,3, Junhou Hui 6, Peng Liu 6, Litao Yang 4, Sheng-Ce Tao 1,2,3
1Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 2State Key Laboratory of Oncogenes and Related Genes, 3School of Biomedical Engineering, Shanghai Jiao Tong University, 4Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 5Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, 6Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

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Developmental Biology

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice
Jeffrey B. Mason 1, Kate C. Parkinson 1, Tracy L. Habermehl 1
1Department of Animal, Dairy and Veterinary Sciences, Center for Integrated BioSystems, School of Veterinary Medicine, Utah State University

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

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Engineering

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting
Amir Behbahanian 1, Nicholas A. Roberts 1
1Mechanical and Aerospace Engineering, Utah State University

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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JoVE Journal

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure
Justin A. Jones 1, Thomas I. Harris 1, Brianne E. Bell 1, Paula F. Oliveira 1
1Department of Biology, Utah State University

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

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Behavior

A Cross-Disciplinary and Multi-Modal Experimental Design for Studying Near-Real-Time Authentic Examination Experiences
Idalis Villanueva 1, Jenefer Husman 3, Darcie Christensen 1, Kate Youmans 1, Md Tarique Khan 1, Paul Vicioso 2, Shawn Lampkins 3, Matthew C. Graham 3
1Department of Engineering Education, College of Engineering, Utah State University, 2Department of Electrical and Computer Engineering, College of Engineering, Utah State University, 3Department of Educational Psychology, College of Education, University of Oregon

An experimental design was developed to investigate the real-time influences of an examination experience to assess the emotional realities students experience in higher education settings and tasks. This design is the result of a cross-disciplinary (e.g., educational psychology, biology, physiology, engineering) and multi-modal (e.g., salivary markers, surveys, electrodermal sensor) approach.

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Behavior

A Modified Lean and Release Technique to Emphasize Response Inhibition and Action Selection in Reactive Balance
David A.E. Bolton 1, Mahmoud Mansour 2
1Department of Kinesiology & Health Science, Utah State University, 2Department of Electrical & Computer Engineering, Utah State University

Here we offer a protocol that allows the user to selectively change affordances and/or constraints on movements that are relevant for recovering balance after postural perturbation.

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JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
Jing Liu 1,2, Dalibor Kurepa 3, Francesco Feletti 4,5, Almudena Alonso-Ojembarrena 6, Jovan Lovrenski 7, Roberto Copetti 8, Erich Sorantin 9, Javier Rodriguez-Fanjul 10, Karishma Katti 3, Andrea Aliverti 4, Huayan Zhang 11,12, Misun Hwang 13, Tsu F. Yeh 14, Cai-Bao Hu 15, Xing Feng 16, Ru-Xin Qiu 1,2, Jing-Han Chi 17, Li-Li Shang 18, Guo-Rong Lyu 19, Shao-Zheng He 20, Yan-Fen Chai 21, Zhan-Jun Qiu 22, Hai-Ying Cao 2,23, Yue-Qiao Gao 1,2, Xiao-Ling Ren 1,2, Guo Guo 1,24, Li Zhang 1,2, Ying Liu 1,2, Wei Fu 1,2, Zu-Lin Lu 1,2, Hong-Lei Li 1,2
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, 5Dipartimento di Diagnostica per Immagini, Ausl della Romagna, S. Maria delle Croci Hospital, 6Neonatal Intensive Care Unit, Puerta del Mar University Hospital, 7Faculty of Medicine, University of Novi Sad, Serbia, Institute for Children and Adolescents Health Care of Vojvodina, 8Emergency Department, University Hospital of Cattinara, 9Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 10Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 11Center for Newborn Care, Guangzhou Women and Children's Medical Center, 12Division of Neonatology, Children's Hospital of Philadelphia, 13Section of Neonatal Imaging, Department of Radiology, Children's Hospital of Philadelphia, 14Maternal Child Health Research institute, Taipei Medical University and China Medical University, 15Intensive Care Unit, Zhejiang Hospital, 16Department of Neonatology, Children's Hospital of Soochow University, 17Department of Neonatology and NICU, Bayi Children's Hospital Affiliated to the Seventh Medical Center of Chinese PLA General Hospital, 18Intensive Care Unit, Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Second Affiliated Hospital of Fujian Medical University, 21Department of Emergency Medicine, Tianjin Medical University General Hospital, 22Department of Emergency and Critical Care Medicine, Affiliated Hospital of Traditional Chinese Medicine, 23Department of Ultrasound, GE Healthcare, 24The Neonatal Intensive Care Unit, Fifth Medical Center of Chinese PLA General Hospital

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

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Bioengineering

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures
Sriram Kumar 1, Ying Liu 1, Rebecca E. Taylor 1,2,3
1Department of Mechanical Engineering, Carnegie Mellon University, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Electrical and Computer Engineering, Carnegie Mellon University

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

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Biology

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances
Houston J. Judd 1,2, Craig Huntzinger 2, Ricardo Ramirez 1, James P. Strange 2,3
1Department of Biology, Utah State University, 2USDA-ARS Pollinating Insect Research Unit, 3Department of Entomology, The Ohio State University

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

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Medicine

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery
Tao Liu *1,2,3,4,5, Xueling Song *1,2,3,4,5, Xiaoying Zheng *1,2,3,4,5, Liying Yan 1,2,3,4,5, Caihong Ma 1,2,3,5, Rong Li 1,2,3,4,5, Jie Yan 1,2,3,4,5, Jie Qiao 1,2,3,4,5
1Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology, 3Key Laboratory of Assisted Reproduction, Ministry of Education, 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, 5Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest, Chinese Academy of Medical Sciences

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

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Genetics

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform
Chengming Xu *1, Riqing Wei *2, Hui Lin 1, Leiyu Deng 1, Li Wang 3, Deyang Li 4, Honghui Den 5, Wensong Qin 1, Ping Wen 1, Ying Liu 1, Yingsong Wu 2, Qiang Ma 2, Jinliang Duan 1
1Centre for Women, Children, and Reproduction, Guangxi Key Laboratory of Metabolic Diseases Research, 2Department of Biopharmaceutics, School of Laboratory Medicine and Biotechnology, Southern Medical University, 3Department of Obstetrics and Gynecology, Chinese PLA General Hospital, 4Clinical Laboratory, Northern Theater Air Force Hospital, 5Guangzhou Darui Reproduction Technology Co., Ltd.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

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Developmental Biology

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte
Laura Adams *1, Ying Liu *1, Barbara Durrant 2, Elena Ruggeri 2, Carly Young 2, Rebecca Krisher 3, Irina Polejaeva 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2Reproductive Sciences, Beckman Center for Conservation Research, San Diego Zoo Wildlife Alliance, 3Genus PLC

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P < 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

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Environment

Big Box Biochar Kiln Operation and Best Practices
Darren J. McAvoy 1
1Utah State University

The Utah Biomass Resources Group (UBRG) has scaled-up simple biochar kilns in an innovative approach to hazardous fuel reduction and biochar production using metal boxes, called Big Box kilns, that allow for in-woods biochar production. This article outlines Big Box biochar kiln operation and best practices.

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Biology

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling
Chase A. Paterson 1, Dillon Weatherston 1, Teren Teeples 1, Elizabeth Vargis 1
1Department of Biological Engineering, Utah State University

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

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