Unos i učinci genskih preinaka posredovanih lentivirusnim vektorom u mišjem mozgu

doctoral thesis

• Title (english): Application and effects of gene modifications mediated by a lentviral vector in the mouse brain
• Author: Laura Skukan
• Mentor: Srećko Gajović (mentor)
• Committee member: Željka Krsnik (predsjednik povjerenstva)
• Committee member: Dubravka Švob Štrac (član povjerenstva)
• Committee member: Nataša Jovanov Milošević (član povjerenstva)
• Committee member: Dinko Mitrečić (član povjerenstva)
• Committee member: Goran Sedmak (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2024-04-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Neuroscience
• Universal decimal classification (UDC): 61 - Medical sciences

Abstract

Unos genetskog materijala i genskih preinaka važan je pristup za proučavanje i liječenje genetskih i drugih bolesti. Lentivirusni vektori ističu se kao dominatan sustav dostave u ciljane stanične linije središnjeg živčanog sustava. Glavni cilj ovog istraživanja bio je utvrditi transdukciju neurona i astrocita za njih specifično dizajniranim lentivirusnim vektorom, procjeniti uzrokuje li dostavljen sustav shRNA utišavanje gena Spry2 te utvrditi učinak utišavanja gena Spry2 nakon izazivanja ishemijskog moždanog udara u mišjem modelu. Potvrđena je uspješna transdukcija stanica lentivirusnim vektorom te je analizom kolokalizacije pokazano da lentivirusni vektori s ovojnicom VSV-G transduciraju neurone dok Tet regulirani lentivirusni vektori sa specifičnim promotorom za astrocite i ovojnicom Mokola transduciraju astrocite. Procjenom aktivacije mikroglije i receptora TLR2 pokazano je da unos lentivirusnog vektora uzrokuje kratkotrajnu aktivaciju upalnog odgovora koja je ograničena na područje unosa. Analizom genske ekspresije potvrđeno je utišavanje gena Spry2 u injektiranim regijama mišjeg mozga. Međutim, njegova smanjena ekspresija nije pridonijela značajnom smanjenju volumena ishemijske lezije i funkcionalnom oporavku nakon ishemijskog moždanog udara. Ovo istraživanje potvrdilo je da su primjenjene strategije dizajna lentivirusnih vektora učinkovit sustav dostave koji omogućava ograničenu transdukciju stanica od interesa i uspješno utišavanje gena Spry2.

Abstract (english)

The delivery of genetic material or gene modification is an important approach for the study and treatment of genetic and other diseases. Lentiviral vectors are a dominant delivery system to target cell lines of the central nervous system. The aim of this study was to determine the transduction of target cells with a specifically designed lentiviral vector for the neuron and astrocyte transduction, to assess whether the delivered shRNA system causes silencing of Spry2, and to examine the effect of Spry2 silencing after ischemic stroke induction in a mouse model. Successful cell transduction with the lentiviral vector was confirmed, and colocalization analysis confirmed that VSV-G pseudotyped lentiviral vectors transduce neurons, while Tet-regulated Mokola pseudotyped lentiviral vector with astrocyte-specific promoter transduce astrocytes. Furthermore, lentiviral vector causes a short-term activation of the inflammatory response, which was restricted near the area of injection site. Silencing of the Spry2 was confirmed in the injected regions of the mouse brain. However, downregulation of Spry2 did not reduced the volume of ischemic lesion and contribute to the functional recovery after ischemia. This study confirmed that the applied design strategies of lentiviral vector are an efficient delivery system that allows restricted transduction of cells of interest and successful Spry2 silencing.

• Keywords: Miševi; Astrociti; Moždani udar; Stanična linija; Neuroni; Mozak; Središnji živčani sustav; Ishemija; RNA; mala interferencija
• Keywords (english): Mice; Astrocytes; Ischemic Stroke; Cell Line; Neurons; Brain; Central Nervous System; Ischemia; RNA; Small Interfering
• Language: croatian
• URN:NBN: urn:nbn:hr:105:277371
• Project: Number: KK.01.1.1.04.0085; Title: Genomsko inženjerstvo i genska regulacija u staničnim linijama i modelnim organizacijama tehnologija CRISPR / Cas9 CasMouse; Title: Genomic engineering and gene regulation in the cell lines model organisms CRISPR / Cas9 CasMouse technology; Acronym: CRISPR/Cas9-CasMouse; Leader: Srećko Gajović; Jurisdiction: Croatia; Funder: EK; Funding stream: EFRR
• Study programme: Title: Neuroscience; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
• Type of resource: Text
• Extent: 120 str.
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-04-17
• Created on: 2024-04-19 10:57:13