magistrsko delo
Sanja Terzić (Author), Sabina Berne (Reviewer), Roman Jerala (Mentor)

Abstract

Protein β-katenin, ki ga kodira gen CTNNB1, sodeluje v signalni poti Wnt in ima pomembno vlogo v zgodnjem nevrološkem razvoju. Pomanjkanje ß-katenina povzroča redek sindrom CTNNB1, za katerega so značilne motnje v kognitivnem in motoričnem razvoju. V magistrski nalogi smo želeli povečati količino citosolnega ß-katenina z zaviranjem delovanja aksina, ki je vpleten v razgradnjo β-katenina s pomočjo destrukcijskega kompleksa. V ta namen smo uporabili protismiselne oligonukleotide (angl. antisense oligonucleotides, ASOs), ki ciljajo vezavna mesta spajalno-izrezovalnega kompleksa na RNA za aksin. Pripravili smo delecijske konstrukte aksina brez izbranih eksonov ter s prenosom western, konfokalno mikroskopijo in pretočno citometrijo preverili njihovo izražanje, ko-lokalizacijo z ß-kateninom ter vpliv na aktivnost ß-katenina v celicah HEK293T. Na podlagi rezultatov smo načrtovali kratke protismiselne oligonukleotide ter testirali, ali lahko sprožijo preskakovanje eksonov na nivoju RNA ter kakšen je njihov vpliv na raven endogenega aksina in ß-katenina v celicah HEK293T. Protismiselni oligonukleotidi so povzročili preskakovanje tarčnih eksonov, znižali raven endogenega aksina ter povišali raven β-katenina v celicah. Po uporabi ASO:eks5 smo potrdili prisotnost dveh različic endogenega aksina. Dobljeni rezultati kažejo na možnost razvoja terapije za zdravljenje sindroma CTNNB1 na osnovi protismiselne tehnologije.

Keywords

sindrom CTNNB1;beta-katenin;destrukcijski kompleks;aksin-1;protismiselni oligonukleotidi;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: KI - National Institute of Chemistry
Publisher: [S. Terzić]
UDC: 601.4:577.21(043.2)
COBISS: 193608707 Link will open in a new window
Views: 74
Downloads: 191
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Other data

Secondary language: English
Secondary title: Inhibition of endogenous ß-catenin inhibitors with designed short antisense oligonucleotides
Secondary abstract: The protein β-catenin, which is encoded by the CTNNB1 gene, is involved in the Wnt signalling pathway and plays an important role in early neurological development. Deficiency of ß-catenin causes a rare CTNNB1 syndrome, which is characterized by impaired cognitive and motor development. Our aim was to increase the amount of cytosolic ß-catenin by inhibiting the activity of Axin, which is involved in the degradation of ß-catenin by the destruction complex. For this purpose, we used antisense oligonucleotides (ASOs) targeting the binding sites of the splicing complex on Axin RNA. Deletion constructs of Axin without selected exons were prepared and their expression, co-localisation with ß-catenin and their influence on ß-catenin activity in HEK293T cells were investigated by western blot, confocal microscopy, and flow cytometry, respectively. We designed short ASOs and tested their ability to induce exon skipping at the RNA level and their effect on endogenous levels of Axin and ß-catenin in HEK293T cells. We demonstrated that exon skipping was successfully induced by ASOs at the RNA level. Treatment with ASOs led to a decrease in endogenous Axin levels and an increase in endogenous β-catenin levels. Two variants of endogenous Axin were detected after treatment with ASO:eks5. These results suggest the possibility of developing a therapy for the treatment of CTNNB1 syndrome based on antisense technology.
Secondary keywords: CTNNB1 syndrome;beta-catenin;destruction complex;Axin-1;antisense oligonucleotides;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za agronomijo
Pages: 1 spletni vir (1 datoteka PDF (XII, 54 str.))
ID: 23306936