Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects the motor neurons of the central nervous system. So far, the disease has been associated with mutations of more than 50 genes, of which the most common mutation is in the C9ORF72 gene (chromosome 9 open reading frame 72). The mutation causes an expansion of GGGGCC (G$_4$C$_2$) repeats in the first intron of the gene, which leads to formation of insoluble RNA foci in the cell nucleus, sequestration of RNA-binding proteins and synthesis of dipeptide repeat proteins (DPR). As part of our research work, we wanted to prepare a reporter system which would help us detect sense and antisense GGGGCC repeats in human cell cultures. If fluorescence were detected in the cell, it would mean that repeats or their RNA transcripts are present in the cell. We used the vector pGint, in which we wanted to insert a different number of sense and antisense GGGGCC repeats and thus create our reporter system. First, we inserted a restriction site for the restriction enzyme HindIII into the pGint vector by site-specific mutagenesis. We then amplified the vectors containing the 8×G$_4$C$_2$, 24×G$_4$C$_2$, 48×G$_4$C$_2$, 8×C$_4$G$_2$, 24×C$_4$G$_2$ and 32×C$_4$G$_2$ repeats and excised the repeats from them. We then tried to insert the repeats into the mutated pGint through the restriction sites for restriction enzymes BamHI and HindIII. Despite difficulties in isolating the repeats from the vector we managed to prepare the pGint vector, which contained sense and antisense hexanucleotide repeats in the intron within the EGFP open reading frame.
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