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Jichi Medical University

15 ARTICLES PUBLISHED IN JoVE

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Biology

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System
Masanori Takahashi 1, Noriko Osumi 1,2
1Division of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, 2The Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

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Biology

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture
Masanori Takahashi 1,2, Sayaka Makino 3, Takako Kikkawa 3, Noriko Osumi 3
1Graduate School of Medicine, Jichi Medical University, 2Division of Biology, Center for Molecular Medicine, Jichi Medical University, 3Department of Developmental Neuroscience, United Center for Advanced Research and Translational Medicine (ART), Tohoku University School of Medicine

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

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Genetics

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR
Keiji Hirai 1, Hiromichi Yoshizawa 2, Toshimi Imai 2, Yusuke Igarashi 2, Ichiro Hirahara 2, Susumu Ookawara 2, Kenichi Ishibashi 3, Yoshiyuki Morishita 1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 2Division of Nephrology, Department of Internal Medicine, Jichi Medical University, 3Department of Medical Physiology, Meiji Pharmaceutical University

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

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Biology

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis
Shinichiro Yokota 1,2, Yoshihiro Ono 2, Toshimasa Nakao 2, Peng Zhang 3, George K. Michalopoulos 4,5, Zahida Khan 3,4,5,6
1Department of Surgery, Jichi Medical University, 2Department of Surgery, University of Pittsburgh School of Medicine, 3Department of Pediatrics, University of Pittsburgh School of Medicine, 4Department of Pathology, University of Pittsburgh School of Medicine, 5Pittsburgh Liver Research Center, University of Pittsburgh, 6McGowan Institute for Regenerative Medicine, University of Pittsburgh

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

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Cancer Research

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment
Rihito Kanamaru 1, Hideyuki Ohzawa 1, Hideyo Miyato 1, Hironori Yamaguchi 1, Yoshinori Hosoya 1, Alan Kawarai Lefor 1, Naohiro Sata 1, Joji Kitayama 1
1Department of Gastrointestinal Surgery, Jichi Medical University

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

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Behavior

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress
Hiroki Sugimoto 1, Kiyoshi Kawakami 1
1Division of Biology, Center for Molecular Medicine, Jichi Medical University

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

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Genetics

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice
Hirofumi Nishizono *1,2,3, Mohamed Darwish *4,5, Hideki Uosaki 6,7, Nanami Masuyama 8,9,10, Motoaki Seki 8,11, Hiroyuki Abe 3, Nozomu Yachie 8,9,10,12,13, Ryohei Yasuda 1
1Max Planck Florida Institute for Neuroscience, 2Life Science Research Center, University of Toyama, 3Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, 4Graduate School of Innovative Life Science, University of Toyama, 5Department of Biochemistry, Faculty of Pharmacy, Cairo University, 6Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 7Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology, Jichi Medical University, 8Synthetic Biology Division, Research Center for Advanced Science and Technology, University of Tokyo, 9Institute for Advanced Biosciences, Keio University, 10Graduate School of Media and Governance, Keio University, 11Department of Molecular Oncology, Graduate School of Medicine, Chiba University, 12Department of Biological Sciences, School of Science, University of Tokyo, 13College of Arts and Sciences, University of Tokyo

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

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Medicine

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model
Akinori Aomatsu 1, Keiji Hirai 1, Hiroki Ishii 1, Katsunori Yanai 1, Shohei Kaneko 1, Yoshiyuki Morishita 1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

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Genetics

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction
Katsunori Yanai 1, Shohei Kaneko 1, Hiroki Ishii 1, Akinori Aomatsu 1,2, Kiyonori Ito 1, Keiji Hirai 1, Susumu Ookawara 1, Kenichi Ishibashi 3, Yoshiyuki Morishita 1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 2Division of Intensive Care Unit, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 3Department of Medical Physiology, Meiji Pharmaceutical University

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

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Immunology and Infection

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice
Shohei Kaneko 1, Katsunori Yanai 1, Hiroki Ishii 1, Akinori Aomatsu 1,2, Kiyonori Ito 1, Keiji Hirai 1, Susumu Ookawara 1, Kenichi Ishibashi 3, Yoshiyuki Morishita 1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 2Division of Intensive Care Unit, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 3Department of Medical Physiology, Meiji Pharmaceutical University

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

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Medicine

Measurement of Tissue Oxygenation Using Near-Infrared Spectroscopy in Patients Undergoing Hemodialysis
Kiyonori Ito *1, Susumu Ookawara *1, Takayuki Uchida 2, Hideyuki Hayasaka 2, Masaya Kofuji 2, Haruhisa Miyazawa 1, Akinori Aomatsu 1, Yuichiro Ueda 1, Keiji Hirai 1, Yoshiyuki Morishita 1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 2Department of clinical engineering, Saitama Medical Center, Jichi Medical University

We present a protocol to measure regional oxygen saturation (rSO2) in hemodialysis (HD) patients by using a near-infrared spectroscopy monitor. The rSO2 value is an index of tissue oxygenation. This noninvasive and real-time monitoring could be useful for confirming changes in organ oxygenation during HD.

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Medicine

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.
Razan E. Ahmed *1, Nawin Chanthra *1, Tatsuya Anzai 1,2, Keiichiro Koiwai 3,4, Tomoki Murakami 4, Hiroaki Suzuki 4, Yutaka Hanazono 1, Hideki Uosaki 1
1Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 2Department of Pediatrics, Jichi Medical University, 3Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 4Department of Precision Mechanics, Faculty of Science and Engineering, Chuo University

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

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Biochemistry

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm
Riho Kurata *1, Masamitsu Harada *2, Jun Nagai *3, Xiaofeng Cui *4, Takayuki Isagawa 5, Hiroaki Semba 6, Yasuhiro Yoshida 7, Norihiko Takeda 8, Koji Maemura 9, Tomo Yonezawa 3
1Education and Research Center for Pharmaceutical Sciences, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, 2Independent Scholar, Tokyo, Japan, 3Division of Functional Genomics and Therapeutic Innovation, Research Center for Advanced Genomics, Graduate School of Biomedical Sciences, Nagasaki University, 4School of Chemistry, Chemical Engineering and Life Sciences, School of Materials and Engineering, Wuhan University of Technology, 5Data Science Center, Jichi Medical University, 6Department of Cardiovascular Medicine, The Cardiovascular Institute, 7Department of Immunology and Parasitology, University of Occupational and Environmental Health, 8Division of Cardiology and Metabolism, Center for Molecular Medicine, Jichi Medical University, 9Department of Cardiovascular Medicine, Graduate School of Biomedical Sciences, Nagasaki University Hospital

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

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Genetics

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment
Katsunori Yanai 1, Shohei Kaneko 1, Hiroki Ishii 1, Akinori Aomatsu 1,2, Kenichi Ishibashi 3, Yoshiyuki Morishita 1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 2Division of Intensive Care Unit, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University, 3Department of Medical Physiology, Meiji Pharmaceutical University

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

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Medicine

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models
Katsunori Yanai *1, Shohei Kaneko 1, Hiroki Ishii 1, Akinori Aomatsu 1, Yoshiyuki Morishita *1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

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