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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.
DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.
A DNA double-strand break (DSB) is the most deleterious form of DNA damage, potentially leading to genome instability, chromosomal rearrangements, cellular senescence, and cell death if not repaired promptly1. Two well-established pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), are recognized for their effectiveness in addressing DNA DSBs2,3. HR is considered an error-free mechanism for DSB repair, utilizing homologous sequences in the sister chromatid as a template to restore the original configuration of the injured DNA molecule
1. Plasmid isolation
To ensure the accuracy of NHEJ and HR analysis, the implementation of a suitable compensation adjustment and gating strategy is necessary. Typically, mCherry fluorescence does not manifest in the GFP detector when using a 530 nm filter. However, in instances of cells exhibiting extremely high GFP expression, the GFP fluorescence may contaminate the mCherry detector when using a 575 nm filter. To address these concerns, negative control, GFP single-color control, and mCherry single-color control samples were used for comp.......
The method described here has been employed in several papers to assess NHEJ efficiency and HR efficiency9,10,11,12,13,14,16,17,18,19. This method is pertinent for elucidating the underly.......
This research was funded by the Natural Science Foundation of Heilongjiang Province of China (YQ2022C036) and the Graduate Innovation Foundation of Qiqihar Medical University (QYYCX2022-06). Figure 1 produced using MedPeer.
....Name | Company | Catalog Number | Comments |
6 cm dishes | BBI | F611202-9001 | |
6 well plates | Corning | 3516 | |
Ampicillin | Beyotime | ST007 | Working concentration: 100 μg/mL |
DH5α Competent Cells | TIANGEN | CB101 | |
DMEM | Hyclone | SH30022.01 | |
DR-GFP | Addgene | 26475 | |
EJ5-GFP | Addgene | 44026 | |
EndoFree Maxi Plasmid kit | TIANGEN | DP117 | alternative endotoxin-free plasmid extraction kit can be used |
FACS tubes | FALCON | 352054 | |
Fetal bovine serum | CLARK | FB25015 | |
Flow cytometer | BD Biosciences | BD FACSCalibur | |
FlowJo V.10.1 | Treestar | alternative analysis software can be used | |
HEK-293T cells | National Infrastructure of Cell Line Resource | 1101HUM-PUMC000091 | |
Lipo3000 | Invitrogen | L3000015 | alternative transfection regents can be used |
PBS | Biosharp | BL601A | |
pCBASceI | Addgene | 26477 | I-SceI expressing plasmid |
PCI2-HA-mCherry | alternative plasmids containing DsRed can be used | ||
Trypsin | Gibco | 25200-056 |
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