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

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.

In this protocol, we describe the extrachromosomal NHEJ and HR assays involving transfection of the disrupted GFP and ISEC1 plasmids into HK2 and N3T cells and subsequent flow cytometry analysis. These non-integrated reporter assays have been employed to assess the NHEJ efficiency and HR efficiency by several labs, including ours. In contrast to the chromosomally integrated approach, this extrachromosomal approach enables swift analysis of the NHEJ or HR efficiency through the utilization of established stable cell lines.

Begin by culturing HEK 293T cells in DMEM medium supplemented with 10%fetal bovine serum. On the day before transfection, seed HEK 293T cells in a six-well plate to achieve approximately 60%confluency the following day. Then, transfect the experimental sample cells in a six-well plate with plasmids for NHEJ or HR assay using a commercial transfection kit.

For fax calibration controls, leave a well of untransfected cells as a negative control. Transfect the cells in a six-well plate with plasmids for GFP, single color control or mCherry single color control. Begin by transfecting HEK 293T cells.

Three days after transfection, remove the media from the wells and wash once with PBS. Add 500 microliters of trypsin to each well. Incubate for five minutes at 37 degrees Celsius to detach all cells from the plate.

Then, add 500 microliters of complete DMEM media to each well and resuspend cells. Transfer all cells from each well into 1.5 milliliter centrifuge tubes. Then, centrifuge for two minutes at 1000 G at room temperature.

Carefully remove the supernatant by pipetting and wash the pellet once with one milliliter PBS. Again, carefully remove the supernatant and resuspend the cells in 500 microliters of PBS. Transfer the cells to fax tubes.

Assay the cells using a flow cytometer. Load the untransfected cell suspension. On the worksheet of flow cytometry, press acquire and adjust the forward and side scatter voltages.

To position the cells in the middle region, gate around this Population R1 to exclude the debris in the lower left quadrant. Change the gate of FL1, FL2. blot as G equals R1.Select the quad gating tool and click on the upper right extreme of the cell population in the FL1, FL2.

blot as G equals R1.Adjust the FL1 FL2 voltages to position the cells in the lower left quadrant. Press pause and abort. Then, load the GFP single color control cells.

Press acquire and adjust compensation to position the GFP positive cells in the lower right quadrant. Press pause and abort. Next, load the mCherry single color control cells.

Press acquire and adjust compensation to position the mCherry positive cells in the upper left quadrant. Press pause and abort. Click acquire on the menu bar and select acquisition and storage.

Under collection criteria, select acquisition will stop when 10, 000 of G1 equals R1 events are counted. Cancel the check mark of setup. Load the sample cells.

Press acquire and wait until 10, 000 G1 equals R1 cells have been counted. Repeat the same for other samples and controls. To begin import all fax files into the analysis dashboard.

Open one of the files to view the forward scatter side scatter plot. Construct a polygon gate to isolate intact cells, avoiding debris in the lower left corner of the plot. Label the subpopulation as intact cells and drag this gate to the all samples bar.

Verify the gating for each sample by using the next sample button. Double click on the intact cells subpopulation of the negative control sample to bring up a new plot. Adjust the plot axis to FL1-H on the x axis to represent GFP and FL2-H on the Y axis representing mCherry.

Next, select the quad gating tool and click on the upper right extreme of the negative cell population. Drag the four rectangular gates to the intact cells bar. Examine each sample of GFP or mCherry single color controls for correct gating using the next sample button.

Navigate to the layout editor. Here, select representative plots and choose copy to layout editor. Select all the representative plots.

Then, double click to open graph definition. Name x axis label as GFP and Y axis label as mCherry. Determine relative DNA repair efficiency by comparing the number of GFP positive cells to the number of mCherry positive cells within the same plot.

A suitable compensation adjustment and gating strategy was implemented to ensure the accuracy of NHEJ and HR analysis. This strategy positioned GFP positive cells in the lower right quadrant and mCherry positive cells in the upper left quadrant. The percentage of GFP positive cells indicated DNA DSB repair and transfection efficiency.

Conversely, the percentage of mCherry positive cells reflected only transfection efficiency. DNA DSB repair efficiency was calculated as the ratio of GFP positive to mCherry positive cells. Additionally, the role of Wiskott-Aldrich syndrome protein and SCAR homologue or WASH protein in DNA repair was demonstrated using the NHEJ assay on shControl and shWASH cells.

The loss of WASH decreased NHEJ efficiency underscoring its role in promoting NHEJ.