data analysis of flow cytometry results from transfected hek-293t cells

对GFP和mCherry报告细胞进行FACS分析，以评估NHEJ和HR效率

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 molecule3. NHEJ, on the other hand, is an error-prone DSB repair pathway that joins the broken DNA ends without relying on any template2.
The NHEJ assay and HR assay are classical methods originally developed in Jasin&#39;s laboratory at Memorial Sloan-Kettering Cancer Center and utilized to quantify the efficiency of NHEJ and HR, respectively4,5,6,7. These assays play a crucial role in investigating the relevant mechanisms and factors associated with NHEJ and HR8,9,10,11,12,13,14. Both assays rely on the implementation of two disrupted GFP reporters, EJ5-GFP and DR-GFP, to monitor the repair of DSBs induced by the I-SceI endonuclease. The EJ5-GFP reporter is employed in the NHEJ assay, while the DR-GFP reporter is utilized in the HR assay. Each reporter is subtly designed so that the I-SceI-induced DSBs can only be repaired by a specific repair pathway to restore a GFP expression cassette4,5.
The NHEJ assay and HR assay can be conducted using either a chromosomally integrated or an extrachromosomal approach15,16. The chromosomally integrated approach necessitates the integration of the disrupted GFP reporters into the genome, allowing the analysis of DSB repair within a chromosomal context6,15. However, this approach requires prolonged cell passaging and is unsuitable for comparative studies involving multiple cell lines due to arbitrary chromosomal integration, introducing an additional confounding factor apart from inherent differences. In this protocol, we describe the extrachromosomal NHEJ assay and HR assays, involving the transient transfection of the disrupted GFP and I-SceI plasmids into HEK-293T cells, followed by flow cytometry analysis (the experiment workflow is shown in Figure 1). These non-integrated reporter assays were originally reported by Jasin&#39;s laboratory to study DNA interstrand cross-links repair16 and have been employed to assess NHEJ efficiency and HR efficiency by several laboratories9,10,11,12,13,14,17,18,19, including ours11. These extrachromosomal approaches facilitate the analysis of DSB repair in comparative studies involving multiple established stable cell lines.

作者聚焦：通过染色体外 NHEJ 检测和 HR 检测解码 DNA 修复

使用基于 GFP 的报告系统分析 HEK-293T 细胞中的非同源末端连接和同源重组效率

DNA double-strand breaks represent the most perilous DNA lesions. In response, cells employ two primary mechanisms for DNA 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 a disrupted green fluorescent protein gene with recognition sites for endonuclease I-SceI for induction of DSBs.The NHEJ assay and the HR assay can be conducted using is a chromosomally-integrated or a extrachromosomal approach. The chromosomally-integrated approach enables the analysis of DSB repair within a chromosomally contest. However, the chromosomally integrated approach requires prolonged cell and is unsuitable for comparative studies involving multiple cell lines.In this protocol, we describe the extrachromosomal NHEJ and HR assays involving transient transfection of the disrupted GFP and I-SceI plasmids into HEK-293T cells and subsequent flow cytometry analysis. These nonintegrated 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.

Data Analysis of Flow Cytometry Results from Transfected HEK-293T Cells

首先，将所有传真文件导入到分析仪表板中。打开其中一个文件以查看前向散射侧散射图。构建一个多边形门来隔离完整的像元，避免在图的左下角出现碎片。将亚群标记为完整细胞，并将此门拖动到所有样本条。使用“下一个样品”按钮验证每个样品的设门。双击阴性对照样本的完整细胞亚群以显示新图。将绘图轴调整为 X 轴上的 FL1-H 表示 GFP，将 Y 轴上的 FL2-H 调整为 mCherry。接下来，选择四门控工具，然后单击负细胞群的右上角。将四个矩形门拖动到完整的单元格条上。使用下一个样品按钮检查 GFP 或 mCherry 单色对照的每个样品，以确保正确设门。导航到布局编辑器。在这里，选择具有代表性的绘图，然后选择复制到布局编辑器。选择所有具有代表性的图，然后双击以打开图形定义。将 X 轴标签命名为 GFP，将 Y 轴标签命名为 mCherry。通过比较同一图中GFP阳性细胞的数量与mCherry阳性细胞的数量来确定相对DNA修复效率。实施了适当的薪酬调整和门控策略，以确保NHEJ和HR分析的准确性。该策略将 GFP 阳性细胞定位在右下象限，将 mCherry 阳性细胞定位在左上象限。GFP阳性细胞的百分比表明DNA DSB修复和转染效率。相反，mCherry 阳性细胞的百分比仅反映了转染效率。DNA DSB 修复效率计算为 GFP 阳性细胞与 mCherry 阳性细胞的比率。此外，使用 shCONTROL 和 shWASH 细胞的 NHEJ 测定证明了 Wiskott-Aldrich 综合征蛋白和 SCAR Humalog 或 WASH 蛋白在 DNA 修复中的作用。水、环境卫生和个人卫生（WASH）的丧失降低了国家卫生和个人卫生项目的效率，突出了其在促进国家卫生和个人卫生方面的作用。

Research

JoVE Journal

Biology

FACS Analysis of GFP and mCherry Reporter Cells to Evaluate NHEJ & HR Efficiency

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

Author Spotlight: Decoding DNA Repair by Extrachromosomal NHEJ Assay and HR Assays

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In ...

科研

生物学

Preparation and Transfection of HEK-293T Cells for FACS Sorting Analysis

Analysis of the GFP-Positive and mCherry-Positive Cells by Flow Cytometry