Konstrukcija vektora za stvaranje transgeničnog miša s bioluminiscentnim i fluorescentnim neuronima

master's thesis

• Title (english): Construction of a vector for the creation of transgenic mouse with bioluminescent and fluorescent neurons
• Author: Magdalena Valenta
• Mentor: Aleksandar Vojta (mentor)
• Mentor: Srećko Gajović (mentor)
• Committee member: Aleksandar Vojta (predsjednik povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Committee member: Romana Gračan (član povjerenstva)
• Committee member: Vlatka Zoldoš (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-09-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Genetički preinačene životinje nužni su modeli za razumijevanje ljudskih bolesti na razini cijelog organizma. Miš s bioluminiscentnim i fluorescentnim neuronima bio bi važan model za proučavanje oštećenja i obnove mozga. Transgenične životinje s bioluminiscentnim i fluorescentnim signalom mogu se koristiti za praćenje molekularnih procesa in vivo, a označene stanice se mogu izolirati i služiti kao model za istraživanja in vitro. U ovom radu je opisana konstrukcija vektora s transgeničnom kazetom koja sadrži mišji promotor teške podjedinice neurofilamenata (NFH, od engl. neurofilament heavy) te gen reporter s bioluminiscentnim i fluorescentnim svojstvima, TurboLuc. Ekspresija reporterskog proteina s konstruiranog plazmida zabilježena je u diferenciranim neuralnim matičnim stanicama. Navedeni plazmid je stoga korišten za dobivanje nove transgenične linije mikroinjekcijom u pronukleus oplođene jajne stanice. Nakon selekcije, nosioci transgena, budući osnivači transgeničnih mišjih linija, snimljeni su optički in vivo te je kod njih utvrđena prisutnost bioluminiscentnog signala. Daljnja karakterizacija novih transgeničnih linija ukazat će je li signal specifičan za terminalno diferencirane neurone. Ovakav mišji model bi bio pogodan za in vitro istraživanje diferencijacije neuralnih matičnih stanica, te in vivo, u praćenju matičnih stanica nakon transplantacije u oštećeno područje mozga.

Abstract (english)

Genetically modified animals are essential models for studying human diseases at the whole-organism level. A mouse with bioluminescent and fluorescent neurons would be a useful model for studies of brain repair after injury. Transgenic animals with fluorescent or bioluminescent signal can be used for in vivo optical imaging. In addition, transgenic animals can be a source of labelled population of cells for in vitro experiments. This thesis describes the construction of a vector with a transgenic cassette composed of mouse NFH (neurofilament heavy) promoter and a reporter gene with bioluminescent and fluorescent properties, TurboLuc. TurboLuc expression from constructed plasmid was detected during neural stem cells differentiation. Subsequently, the constructed plasmid was used for generation of novel transgenic mouse line by pronuclear microinjection. The new-born pups were genetically screened and transgenic carriers were selected. Bioluminescent signal was detected in transgenic animals using in vivo optical imaging. Future experiments would indicate if the signal from the reporter is specific for terminally differentiated neurons. The novel transgenic mouse could be appropriate for the research of differentiation of neural stem cells, both in vitro and in vivo. This would provide an insight in the effect of neural stem cells transplantation in the disease-affected brains.

• Keywords: neurofilamenti; Nefh; TurbLuc; luciferaza; Luc2; TurboFP635; neuralne matične stanice
• Keywords (english): neurofilaments; neurofilament heavy; Nefh; TurboLuc; luciferase; Luc2; TurboFP635; neural stem cells
• Language: croatian
• URN:NBN: urn:nbn:hr:217:115962
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-10-29 11:44:50