Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

Lu-Ping Zhang; Yong-Hong Nie; Tuo Tang; Ai-Xue Zheng; Xian Hong; Tao Wang

doi:10.3791/66501

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells U...

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Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

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09:29 min

February 2, 2024

DOI: 10.3791/66501-v

Lu-Ping Zhang¹, Yong-Hong Nie¹, Tuo Tang¹, Ai-Xue Zheng¹, Xian Hong¹, Tao Wang¹

¹Laboratory of Protein Structure and Function, Institute of Medicine and Pharmacy,Qiqihar Medical University

Overview

This study outlines protocols for extrachromosomal nonhomologous end joining (NHEJ) and homologous recombination (HR) assays to assess the efficiency of DNA double strand break repairs in HEK-293T cells. The assay techniques leverage the use of plasmids, enabling swift analysis of DNA repair mechanisms in a controlled environment.

Key Study Components

Research Area

DNA double strand break repair mechanisms
Cellular response to DNA damage
Genetic assays for repair efficiency

Background

NHEJ and HR are critical for repairing DNA lesions
Efficiency quantification assists in understanding DNA repair dynamics
Extrachromosomal assays allow faster analysis than integrated methods

Methods Used

Extrachromosomal NHEJ and HR assays
HEK-293T cells as the model system
Flow cytometry for quantifying repair efficiency

Main Results

The study demonstrates the successful application of non-integrated reporter assays
Efficiency of NHEJ and HR can be quantitatively compared
Impact of specific proteins on repair efficiency was noted

Conclusions

The protocols establish reliable methods to evaluate DNA repair efficiency
Findings provide insights into genetic mechanisms involved in DNA repair

Frequently Asked Questions

What are NHEJ and HR?

NHEJ (nonhomologous end joining) and HR (homologous recombination) are two primary mechanisms that cells use to repair double strand breaks in DNA.

Why use HEK-293T cells?

HEK-293T cells are widely used in research due to their high transfection efficiency and ability to grow rapidly in culture.

What is the advantage of extrachromosomal assays?

Extrachromosomal assays allow for quicker analysis of repair efficiency compared to chromosomally integrated approaches, making them suitable for comparative studies.

How are the efficiencies of NHEJ and HR quantified?

The efficiencies are quantified through flow cytometry, measuring the ratio of reporter gene expression (such as GFP) relative to control markers (e.g., mCherry).

What role does the WASH protein play in DNA repair?

The study suggests that the WASH protein is involved in modulating NHEJ efficiency, highlighting its significance in the DNA repair process.

Can these methods be applied to other cell types?

Yes, while this study focuses on HEK-293T cells, the protocols can be adapted for other cell lines to study DNA repair mechanisms.

What are fluorescence reporter genes used for?

Fluorescence reporter genes serve as indicators of successful DNA repair events, enabling quantification through fluorescence-based methods.

This protocol describes an extrachromosomal nonhomologous end joining (NHEJ) assay and homologous recombination (HR) assay to quantify the efficiency of NHEJ and HR in HEK-293T cells.

Double strand breaks represent the most paralyzing lesions. In response, cells employ two primary mechanisms for the double strand break repair, NHEJ and HR.Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with them. The NHEJ assay and the HR assay are established methods used to measure the efficiency of NHEJ and HR.These methods rely on meticulously designed plasmids that contain disruptive fluorescence reporter genes with recognition sites for nucleases, a cycle for induction of DSBS.

The NHEJ assay and the HR assay can be conducted using a chromosomally integrated or architectural chromosomal approach. The chromosomally integrated approach enables the analysis of DSB repair within a chromosomally context. However, the chromosomally integrated approach requires prolonged cell passage and is unsuitable for comparative studies involving multiple cell lines.

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