diplomsko delo
Eva Gartner (Author), Boris Rogelj (Mentor)

Abstract

Amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD) sta nevrodegenerativni bolezni, za kateri je značilno postopno slabšanje simptomov, ki neizogibno vodijo v smrt. Razlog za razvoj so lahko mutacije v več potencialnih genih. Kot enega izmed ključnih genov za razvoj ALS/FTD se smatra C9orf72. Mutacija se pojavi znotraj prvega introna, v katerem se nahajajo heksanukleotidne ponovitve GGGGCC. V normalnih pogojih jih je manj kot 30, ob mutaciji pa se število lahko poveča tudi na več tisoč. Ena glavnih hipotez, ki ponuja razlago za patološke vplive podaljšanih ponovitev v genu C9orf72, je akumulacija toksičnih agregatov proteinov z dipeptidnimi ponovitvami (DPR). Poznamo pet različnih proteinov DPR: poli(GA), poli(GR), poli(PA), poli(PR) in poli(GP). Nastanejo pri posebni obliki translacije, ki poteka neodvisno od začetnega kodona AUG. Pripravili smo pet celičnih linij, ki stabilno izražajo proteine DPR s 125 ponovitvami. Iz vnaprej pripravljenih plazmidnih vektorjev smo z restrikcijo izrezali zapise za proteine DPR in jih preklonirali v ekspresijski plazmidni vektor pcDNA5 FRT TO mScarlet-I Myc stop. Želeli smo, da se proteini DPR izražajo skupaj s fluorescenčnim markerjem mScarlet. Ker vsi proteini DPR niso bili v enakem bralnem okvirju kot marker, smo bralni okvir osnovnega ekspresijskega vektorja premaknili za enega oz. dva nukleotida. Z dobljenimi konstrukti smo transficirali Flp-in celično linijo HEK293T. Flp-in sistem nam omogoča integracijo zapisov za proteine DPR v genom celic preko FRT mest, ki se nahajata na osnovnem plazmidu in v genomu celic. Po transfekciji in selekciji celic smo posamezne kolonije pripravili za mikroskopiranje in s fluorescenčno mikroskopijo preverili uspešnost izražanja proteinov DPR. S pomočjo fluorescenčnega markerja mScarlet smo dokazali izražanje proteinov DPR v celici. Ker smo pred mikroskopiranjem dodali tudi barvilo DAPI, smo lahko opazovali tudi lokalizacijo proteinov v celici.

Keywords

amiotrofična lateralna skleroza;ALS;frontotemporalna demenca;FTD;proteini z dipeptidnimi ponovitvami;C9orf72;HEK293T;sistem Flp-in;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [E. Gartner]
UDC: 577.112:616.8-009.5(043.2)
COBISS: 82543363 Link will open in a new window
Views: 240
Downloads: 48
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Other data

Secondary language: English
Secondary title: Preparation of cell models with stable expression of dipeptide repeat proteins (DPR) and evaluation of their role
Secondary abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are both neurodegenerative diseases, characterized by gradual progressing symptoms, that inevitably lead to death. Both are connected to mutations in several potential genes. C9orf72 is thought to be one of the key genes involved in development of ALS/FTD. Mutation occurs in the hexanucleotide (GGGGCC) repeats region inside the first intron. Normally there are about 30 repeats, but mutation can raise that number to more than thousand. One of the main hypotheses explaining the pathological effect of extended hexanucleotide repeats is accumulation of toxic proteins with dipeptide repeats (DPR). There are five proteins DPR: poly(GA), poly(GR), poly(PA), poly(PR), poly(GP), which are produced by translation that is independent of the start codon AUG. We prepared five cell lines that would stably express proteins DPR with 125 repeats. We used restriction to cut fragments of DNA that code for proteins DPR from previously prepared plasmid vectors and cloned them in expression plasmid vector pcDNA5 FRT TO mScarlet-I Myc stop. We wanted proteins DPR to cotranslate with fluorescent marker mScarlet, but not all proteins were in the same reading frame as the marker. For that reason we moved the reading frame of the expression vector for one and for two nucleotides. We transfected Flp-in HEK293T cell line with these constructs. Flp-in system allows us integration of DNA in the genome via Flp recombinase-mediated DNA recombination at the FRT sites, located on expression vector and cell genome. After transfection and cell selection we prepared selected colonies for microscopy and confirmed successful expression of proteins DPR with fluorescent microscopy. We also added DAPI so we could observe DPR localization in cells.
Secondary keywords: dipeptide repeat proteins;ALS;FTD;C9orf72;HEK293T;Flp-in system;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 41 str.
ID: 13359960