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. 2021 Jul-Sep;13(3):122-125.
doi: 10.32607/actanaturae.11430.

A Method for Assessing the Efficiency of the Nucleotide Excision Repair System Ex Vivo

A A Popov  1 , K E Orishchenko  2   3 , K N Naumenko  1 , A N Evdokimov  1 , I O Petruseva  1 , O I Lavrik  1   3
Affiliations

A Method for Assessing the Efficiency of the Nucleotide Excision Repair System Ex Vivo

A A Popov et al. Acta Naturae. 2021 Jul-Sep.

Abstract

The nucleotide excision repair (NER) is one of the main repair systems present in the cells of living organisms. It is responsible for the removal of a wide range of bulky DNA lesions. We succeeded in developing a method for assessing the efficiency of NER in the cell (ex vivo), which is a method based on the recovery of TagRFP fluorescent protein production through repair of the damage that blocks the expression of the appropriate gene. Our constructed plasmids containing bulky nFlu or nAnt lesions near the tagrfp gene promoter were shown to undergo repair in eukaryotic cells (HEK 293T) and that they can be used to analyze the efficiency of NER ex vivo. A comparative analysis of the time dependence of fluorescent cells accumulation after transfection with nFlu- and nAnt-DNA revealed that there are differences in how efficient their repair by the NER system of HEK 293T cells can be. The method can be used to assess the cell repair status and the repair efficiency of different structural damages.

Keywords: DNA damages; ex vivo methods; nucleotide excision repair.

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Figures

Fig. 1
Fig. 1
Schematic of a method for assessing the NER system efficiency ex vivo
Fig. 2
Fig. 2
TagRFP expression in HEK 293T cells transfected with plasmid DNAs. The images were created by overlay of fluorescence and phase-contrast images in ImageJ. Plasmid DNA substrates are shown on left; time after cell transfection is shown on top
Fig. 3
Fig. 3
3. Analysis of the NER efficiency of plasmid DNAs ex vivo in HEK 293T cells. (A) – the number of fluorescent cells (%) over time after transfection with plasmid DNAs; (B) – a representative diagram demonstrating the differences in the quantities of fluorescent cells transfected with nFlu- or nAnt-DNA 12 h and 16 h after transfection. The confidence levels are *p < 0.01 and **p < 0.05
See this image and copyright information in PMC

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