Czech Academy of Sciences 12 articles published in JoVE Bioengineering Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care Lyubomyr Lytvynchuk*1,2, Zbynek Stranak*3, Hana Studenovska4, David Rais4, Štěpán Popelka4, Lucie Tichotová5,6, Yaroslav Nemesh5,6, Anastasiia Kolesnikova5,6, Ruslan Nyshchuk5,6, Anna Brymová5,6, Zdeňka Ellederová5, Jana Čížková5, Jana Juhásová5, Štefan Juhás5, Pavla Jendelová7, Richárd Nagymihály8, Igor Kozak9, Slaven Erceg10, Susanne Binder11, Brigitte Müller1, Knut Stieger1, Jan Motlik5, Goran Petrovski*8, Taras Ardan*5 1Eye Clinic, Department of Ophthalmology, University Hospital Giessen and Marburg GmbH, 2Karl Landsteiner Institute for Retinal Research and Imaging, 3Department of Ophthalmology, University Hospital Kralovske Vinohrady and Third Faculty of Medicine, Charles University in Prague, 4Institute of Macromolecular Chemistry, Czech Academy of Sciences, 5Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 6Department of Cell Biology, Faculty of Science, Charles University, 7Institute of Experimental Medicine, Czech Academy of Sciences, 8Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 9Moorfields Eye Hospitals UAE, 10Stem Cell Therapies in Neurodegenerative Diseases Lab, Research Center “Principe Felipe”, 11Eye Center Donaustadt, Department of Ophthalmology, Sigmund Freud University The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs. Neuroscience Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection Adriana Roithová1,2, David Staněk1 1Laboratory of RNA Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, 2Department of Genetics and Microbiology, Faculty of Science, Charles University Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell. Biochemistry Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei Ondřej Gahura1, Alena Zíková1,2 1Biology Centre, Czech Academy of Science, Institute of Parasitology, 2Faculty of Science, University of South Bohemia This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies. Biochemistry Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis Tomáš Zavřel1, Dominik Chmelík1,2, Maria A. Sinetova3, Jan Červený1 1Department of Adaptive Biotechnologies, Global Change Research Institute, Czech Academy of Sciences, 2Department of Plant Physiology, Faculty of Science, Masaryk University, 3Laboratory of Intracellular Regulation, Institute of Plant Physiology, Russian Academy of Sciences Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually. Biochemistry Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion Miroslava Stastna1,2,3, Amandine Thomas1, Juliana Germano1, Somayeh Pourpirali1, Jennifer E. Van Eyk1,2, Roberta A. Gottlieb1 1The Smidt Heart Institute, Cedars-Sinai Medical Center, 2Advanced Clinical Biosystems Research Institute, Cedars-Sinai Medical Center, 3Institute of Analytical Chemistry of the Czech Academy of Sciences Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome. Behavior Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients Lindsay Canniff1, Miri Dainson2, Analía V. López3, Mark E. Hauber2, Tomáš Grim4, Peter Samaš5, Daniel Hanley1 1Department of Biology, Long Island University-Post, 2Department of Animal Biology, School of Integrative Biology, University of Illinois, 3Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 4Department of Zoology and Laboratory of Ornithology, Palacký University, 5Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments. Biology Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches Jia-Hsin Huang1, Yun Liu1, Yu-Hsien Lin1,2,3, Xavier Belles4, How-Jing Lee1 1Department of Entomology, National Taiwan University, 2Biology Centre, Institute of Entomology, Czech Academy of Sciences, 3Faculty of Science, University of South Bohemia, 4Institute of Evolutionary Biology, CSIC-UPF This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes. Biology Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions Soňa Legartová*1, Jana Suchánková*1, Jana Krejčí1, Alena Kovaříková1, Eva Bártová1 1Institute of Biophysics of the Czech Academy of Sciences Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis. Chemistry Aerosol-assisted Chemical Vapor Deposition of Metal Oxide Structures: Zinc Oxide Rods Stella Vallejos1,2, Naděžda Pizúrová3, Jan Čechal4, Isabel Gràcia1, Carles Cané1 1Instituto de Microelectrónica de Barcelona (IMB-CNM, CSIC), 2SIX Research Centre, Brno University of Technology, 3Institute of Physics of Material, Academy of Science of Czech Republic, 4Institute of Physical Engineering and Central European Institute of Technology, Brno University of Technology Columnar zinc oxide structures in the form of rods are synthesized via aerosol-assisted chemical vapor deposition without the use of pre-deposited catalyst-seed particles. This method is scalable and compatible with various substrates based on either silicon, quartz or polymers. Developmental Biology Live Imaging of Mouse Secondary Palate Fusion Seungil Kim1, Jan Prochazka2, Jeffrey O. Bush1 1Department of Cell and Tissue Biology, Program in Craniofacial Biology and Institute for Human Genetics, University of California San Francisco, 2Laboratory of Transgenic Models Diseases, Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems. Neuroscience Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice Takahiro Tadokoro1, Atsushi Miyanohara1, Michael Navarro1, Kota Kamizato1, Stefan Juhas2, Jana Juhasova2, Silvia Marsala1, Oleksandr Platoshyn1, Erik Curtis3, Brandon Gabel3, Joseph Ciacci3, Nada Lukacova4, Katarina Bimbova4, Martin Marsala1,4 1Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, 2Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 3Department of Neurosurgery, University of California, San Diego, 4Institute of Neurobiology, Slovak Academy of Sciences The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice. Chemistry Synthesis of Zeolites Using the ADOR (Assembly-Disassembly-Organization-Reassembly) Route Paul S. Wheatley1, Jiří Čejka2, Russell E. Morris1 1EaStChem School of Chemistry, The University of St Andrews, 2J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic A protocol for the preparation of novel zeolites by the ADOR (Assembly-Disassembly-Organization-Reassembly) synthetic route is presented.
