The regulation of gene expression can be influenced at the level of transcription, maturation of the primary transcript pre-mRNA and translation. In this master's thesis, we present the approach of regulating gene expression at the level of translation using antisense oligonucleotides (ASO). We designed ASO that bind specifically to sequences in the 5' UTR region, which have potentially inhibitory effects on translation initiation due to the formation of secondary structures. ASO binding can disrupt secondary structures and facilitate translation initiation and consequently increase gene expression. We tested this approach on the CTNNB1 gene, which encodes the protein β-catenin, that has an important structural and signaling role in the cell. The presence of a mutation in one of the alleles of the CTNNB1 gene, due to the reduced expression of β-catenin, causes CTNNB1 syndrome, therefore the application of ASO to increase the expression of β-catenin has the potential for therapeutic use. Using the TopFlash/FopFlash reporter, we showed by measuring luminescence that transfection of ASO into HEK293T cells increases activation of the Wnt signaling pathway, in which β-catenin is involved. After transfection of ASO into HEK293T, SH-SY5Y and iPSC cell lines, we showed by immunodetection of proteins on the membrane that the addition of target ASO leads to an increase in the amount of β-catenin in the cells of all tested cell lines. As we have shown that the use of designed ASO increases the expression and at the same time achieves increased activity of β-catenin, this approach combined with an appropriate delivery method would be promising for clinical use.
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