Journal: Molecular Therapy. Nucleic Acids

Article Title: Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells

doi: 10.1016/j.omtn.2019.12.015

Figure Lengend Snippet: Gene Correction Strategy Using Repair-PPRHs (A) Structure of a repair-PPRH, consisting of a hairpin core linked to a repair domain. The core is formed by two polypurine domains linked by five thymidines, which are bound intramolecularly by reverse-Hoogsteen bonds that bind to its polypyrimidine target sequence in the dsDNA via Watson-Crick bonds. The repair domain is a sequence homologous to the DNA sequence to be repaired but containing the corrected nucleotide instead of the mutation. Mutation in the dsDNA is represented in red, whereas the correct nucleotide contained in the repair-PPRH is shown in green. (B) Scheme of a long-distance repair-PPRH containing a hairpin core that binds to a polypyrimidine target sequence away from the location of the mutation. This hairpin core is linked to a repair domain containing the wild-type nucleotide (green) by five additional thymidines (5T). Mutation in the DNA is represented in red. (C) General procedure for the targeted correction using repair-PPRHs of the adenosyl phosphoribosyl transferase ( aprt ) gene in a collection of aprt -deficient CHO cell lines. The approach involved transfection and selection (+AAT) of the repaired cells followed by sequencing analyses and determination of mRNA levels and enzymatic activity.

Article Snippet: An APRT mRNA TaqMan probe (assay ID: Cg04465038_g1) was used to determine the mRNA expression of the aprt gene.

Techniques: Sequencing, Mutagenesis, Transfection, Selection, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells

doi: 10.1016/j.omtn.2019.12.015

Figure Lengend Snippet: APRT mRNA and Enzymatic Analyses (A–C) APRT mRNA levels of mutants S23 (A), S62 (B), and S1 (C) and their repaired counterparts obtained after transfection with the corresponding repair-PPRHs and subsequent selection are shown. In (C), also represented are the aprt mRNA levels in three clones repaired using the long-distance repair-PPRH (LD-HpS1E2rep). APRT mRNA levels were determined by qRT-PCR and normalized with TBP mRNA. Data are plotted relative to the wild-type cell line D422. (D–F) APRT enzymatic activity was determined for mutants S23 (D), S62 (E), and S1 (F) and their repaired counterparts obtained after transfection with the corresponding repair-PPRHs and subsequent selection. In (F), also shown are the APRT enzymatic activity levels of three clones repaired with the long-distance repair-PPRH (LD-HpS1E2rep). The D422 wild-type cell line was included in the determination as the positive control. Data are represented as mU of APRT enzyme divided by the mg of total protein extract. Error bars represent the standard error of the mean of three experiments. Statistical analysis was performed comparing the mean value of each clone with the mean value of the mutant sample. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: An APRT mRNA TaqMan probe (assay ID: Cg04465038_g1) was used to determine the mRNA expression of the aprt gene.

Techniques: Transfection, Selection, Clone Assay, Quantitative RT-PCR, Activity Assay, Positive Control, Mutagenesis