preparation and transfection of hek-293t cells for facs sorting analysis

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.

Voorbereiding en transfectie van HEK-293T-cellen voor FACS-sorteeranalyse

Begin met het kweken van HEK 293 T-cellen in DMEM-medium, aangevuld met 10% foetaal runderserum. Zaai op de dag voor transfectie HEK 293 T-cellen in een plaat met zes putjes om de volgende dag ongeveer 60% confluentie te bereiken. Transfecteer vervolgens de experimentele monstercellen in een plaat met zes putjes met plasmiden voor NHEJ- of HR-assay met behulp van een commerciële transfectiekit.Laat voor facs-kalibratiecontroles een putje met niet-getransfecteerde cellen over als negatieve controle. Transfecteer de cellen in een plaat met zes putjes met plasmiden voor GFP, enkelvoudige kleurcontrole of mCherry, enkelvoudige kleurcontrole.

preparation-transfection-hek-293t-cells-for-facs-sorting

Research

JoVE Journal

Biology

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

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

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