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University of Ljubljana

11 ARTICLES PUBLISHED IN JoVE

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Biology

Electrochemotherapy of Tumours
Gregor Sersa 1, Damijan Miklavcic 2
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.

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Biology

Measuring the Induced Membrane Voltage with Di-8-ANEPPS
Gorazd Pucihar 1, Tadej Kotnik 1, Damijan Miklavčič 1
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.

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Biology

Cell Electrofusion Visualized with Fluorescence Microscopy
Katja Trontelj 1, Marko Ušaj 1, Damijan Miklavčič 1
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.

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Biology

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Julian Konig 1, Kathi Zarnack 2, Gregor Rot 3, Tomaz Curk 3, Melis Kayikci 1, Blaz Zupan 3, Daniel J. Turner 4, Nicholas M. Luscombe 2, Jernej Ule 1
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.

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Medicine

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro
Mojca Pavlin 1, Saša Haberl 2, Matej Reberšek 2, Damijan Miklavčič 2, Maša Kandušer 2
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.

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Cancer Research

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats
Natasa Tozon 1, Ursa Lampreht Tratar 2, Katarina Znidar 3, Gregor Sersa 2, Justin Teissie 4,5, Maja Cemazar 2,3
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.

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Genetics

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Polona Šafarič Tepeš 1,2, Raffaella Sordella 1
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.

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Genetics

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis
Tadej Pajič 1,2, Tanja Belčič Mikič 1, Helena Podgornik 1,3, Jurka Klun 1, Sandra Šućurović 1, Samo Zver 1, Matjaz Sever 1
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.

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Cancer Research

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis
Nataša Resnik 1, Rok Romih 1, Mateja Erdani Kreft 1, Samo Hudoklin 1
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.

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Environment

Dendrochronological Dating and Provenancing of String Instruments
Katarina Čufar 1, Blaž Demšar 2, Micha Beuting 2, Angela Balzano 1, Nina Škrk 1, Luka Krže 1, Maks Merela 1
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.

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Bioengineering

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells
Tina Batista Napotnik 1, Bor Kos 1, Lea Rems 1
1Faculty of Electrical Engineering, University of Ljubljana

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

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