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

FACS-analyse van GFP- en mCherry Reporter-cellen om de NHEJ- en HR-efficiëntie te evalueren

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.

Auteur in de schijnwerpers: DNA-reparatie decoderen door extrachromosomale NHEJ-test en HR-assays

Analyse van niet-homologe eindverbindingen en homologe recombinatie-efficiëntie in HEK-293T-cellen met behulp van op GFP gebaseerde reportersystemen

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

Importeer om te beginnen alle faxbestanden in het analysedashboard. Open een van de bestanden om het spreidingsdiagram aan de voorwaartse verstrooiingszijde te bekijken. Bouw een polygoonpoort om intacte cellen te isoleren en vermijd puin in de linkerbenedenhoek van het perceel.Label de subpopulatie als intacte cellen en sleep deze poort naar de balk met alle steekproeven. Controleer de poort voor elk monster met behulp van de knop voor het volgende monster. Dubbelklik op de subpopulatie van intacte cellen van het negatieve controlemonster om een nieuwe plot te openen.Pas de plotas aan naar FL1-H op de X-as om GFP weer te geven en FL2-H op de Y-as die mCherry vertegenwoordigt. Selecteer vervolgens de quad gating-tool en klik op het uiterste rechtsboven van de negatieve celpopulatie. Sleep de vier rechthoekige poorten naar de balk met intacte cellen.Onderzoek elk monster van GFP- of mCherry-besturingselementen in één kleur op de juiste gating met behulp van de volgende monsterknop. Navigeer naar de lay-outeditor. Selecteer hier representatieve plots en kies kopiëren naar lay-outeditor.Selecteer alle representatieve plots en dubbelklik vervolgens om de grafiekdefinitie te openen. Noem het label van de X-as als GFP en het label van de Y-as als mCherry. Bepaal de relatieve DNA-herstelefficiëntie door het aantal GFP-positieve cellen te vergelijken met het aantal mCherry-positieve cellen in dezelfde plot.Er werd een geschikte strategie voor het aanpassen van vergoedingen en poorten geïmplementeerd om de nauwkeurigheid van de NHEJ- en HR-analyse te waarborgen. Deze strategie positioneerde GFP-positieve cellen in het kwadrant rechtsonder en mCherry-positieve cellen in het kwadrant linksboven. Het percentage GFP-positieve cellen gaf DNA-DSB-herstel en transfectie-efficiëntie aan.Omgekeerd weerspiegelde het percentage mCherry-positieve cellen alleen de transfectie-efficiëntie. De efficiëntie van DNA DSB-herstel werd berekend als de verhouding tussen GFP-positieve en mCherry-positieve cellen. Bovendien werd de rol van het eiwit van het Wiskott-Aldrich-syndroom en SCAR Humalog of WASH-eiwit bij DNA-herstel aangetoond met behulp van de NHEJ-test op shCONTROL- en shWASH-cellen.Het verlies van WASH verminderde de efficiëntie van NHEJ, wat de rol ervan bij het promoten van NHEJ onderstreept.

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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 ...

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

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