University of Ljubljana View Institution's Website 10 articles published in JoVE Environment Dendrochronological Dating and Provenancing of String Instruments Katarina Čufar1, Blaž Demšar2, Micha Beuting2, Angela Balzano1, Nina Škrk1, Luka Krže1, Maks Merela1 1Department of Wood Science and Technology, Biotechnical Faculty, University of Ljubljana, 2Independent Scholar The dendrochronological analysis of a stringed instrument requires inspecting the top plate, measuring the tree ring widths, establishing the chronology of the instrument, and dating through determining the end date-the year of the formation of the most recent tree ring. Cancer Research Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis Nataša Resnik1, Rok Romih1, Mateja Erdani Kreft1, Samo Hudoklin1 1Faculty of Medicine, Institute of Cell Biology, University of Ljubljana We present a protocol for the isolation and freeze-fracturing of extracellular vesicles (EVs) originating from cancerous urothelial cells. The freeze-fracture technique revealed the EVs' diameter and shape and-as a unique feature-the internal organization of the EV membranes. These are of immense importance in understanding how EVs interact with the recipient membranes. Genetics A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization Polona Šafarič Tepeš1,2, Raffaella Sordella1 1Cold Spring Harbor Laboratory, 2Faculty of Pharmacy, University of Ljubljana Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models. Genetics Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis Tadej Pajič1,2, Tanja Belčič Mikič1, Helena Podgornik1,3, Jurka Klun1, Sandra Šućurović1, Samo Zver1, Matjaz Sever1 1Department of Hematology, University Medical Centre Ljubljana, 2Clinical Institute for Genomic Medicine, University Medical Centre Ljubljana, 3Faculty of Pharmacy, University of Ljubljana High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified. Cancer Research Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats Natasa Tozon1, Ursa Lampreht Tratar2, Katarina Znidar3, Gregor Sersa2, Justin Teissie4,5, Maja Cemazar2,3 1Clinic for Surgery and Small Animals, Veterinary Faculty, University of Ljubljana, 2Department of Experimental Oncology, Institute of Oncology Ljubljana, 3Faculty of Health Sciences, University of Primorska, 4IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS, 5IPBS (Institut de Pharmacologie et de Biologie Structurale), Université de Toulouse Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors. Medicine Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro Mojca Pavlin1, Saša Haberl2, Matej Reberšek2, Damijan Miklavčič2, Maša Kandušer2 1Department of Fundamentals of Electrical Engineering, Mathematics and Physics, University of Ljubljana, 2Department of Biomedical Engineering, University of Ljubljana Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell. Biology iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution Julian Konig1, Kathi Zarnack2, Gregor Rot3, Tomaz Curk3, Melis Kayikci1, Blaz Zupan3, Daniel J. Turner4, Nicholas M. Luscombe2, Jernej Ule1 1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein. Biology Cell Electrofusion Visualized with Fluorescence Microscopy Katja Trontelj1, Marko Ušaj1, Damijan Miklavčič1 1Faculty of Electrical Engineering, University of Ljubljana In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy. Biology Measuring the Induced Membrane Voltage with Di-8-ANEPPS Gorazd Pucihar1, Tadej Kotnik1, Damijan Miklavčič1 1Department of Biomedical Engineering, Faculty of Electrical Engineering, University of Ljubljana External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (ΔΦ). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the ΔΦ noninvasively. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS. Biology Electrochemotherapy of Tumours Gregor Sersa1, Damijan Miklavcic2 1Department of Experimental Oncology, Institute of Oncology Ljubljana, 2Faculty of Electrical Engineering, University of Ljubljana Electrochemotherapy is a combined use of certain chemotherapeutic drugs such as cisplatin and bleomycin and electric pulses applied to the treated tumour nodule. We present the clinical protocol of electrochemotherapy for treatmen of subcutaneous and cutaneous metastases of melanoma.