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Guangxi Key Laboratory of Metabolic Diseases Research

6 ARTICLES PUBLISHED IN JoVE

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.

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 Copetti³, Erich Sorantin⁴, Jovan Lovrenski⁵, Javier Rodriguez-Fanjul⁶, Dalibor Kurepa⁷, Xing Feng⁸, Luigi Cattaross⁹, Huayan Zhang^10,11, Misun Hwang¹², Tsu F. Yeh^13,14, Yisrael Lipener⁷, Abhay Lodha¹⁵, Jia-Qin Wang¹⁶, Hai-Ying Cao^2,17, Cai-Bao Hu^2,18, Guo-Rong Lyu¹⁹, Xin-Ru Qiu^1,2, Li-Qun Jia²⁰, Xiao-Man Wang²⁰, 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 Wang²¹, Zu-Lin Lu^1,2, Hua-Wei Wang⁸, Li-Li Shang²²

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.

JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure

Jing Liu^1,2, Dalibor Kurepa³, Francesco Feletti^4,5, Almudena Alonso-Ojembarrena⁶, Jovan Lovrenski⁷, Roberto Copetti⁸, Erich Sorantin⁹, Javier Rodriguez-Fanjul¹⁰, Karishma Katti³, Andrea Aliverti⁴, Huayan Zhang^11,12, Misun Hwang¹³, Tsu F. Yeh¹⁴, Cai-Bao Hu¹⁵, Xing Feng¹⁶, Ru-Xin Qiu^1,2, Jing-Han Chi¹⁷, Li-Li Shang¹⁸, Guo-Rong Lyu¹⁹, Shao-Zheng He²⁰, Yan-Fen Chai²¹, Zhan-Jun Qiu²², 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.

Bioengineering

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Sriram Kumar¹, Ying Liu¹, 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.

Genetics

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

Chengming Xu^*1, Riqing Wei^*2, Hui Lin¹, Leiyu Deng¹, Li Wang³, Deyang Li⁴, Honghui Den⁵, Wensong Qin¹, Ping Wen¹, Ying Liu¹, Yingsong Wu², Qiang Ma², Jinliang Duan¹

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.

Developmental Biology

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

Laura Adams^*1, Ying Liu^*1, Barbara Durrant², Elena Ruggeri², Carly Young², Rebecca Krisher³, Irina Polejaeva¹

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.