Name: Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells (Video) | JoVE Journal
Uploaded: 2010-09-08T17:00:00.000+00:00
Duration: 13 min 10 s
Description: Learn GFP-based fluorescence assays to quantify homologous recombination and nonhomologous end joining in mammalian cells. This method measures DNA double-strand break repair efficiency and accuracy.

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The original GFP-Pem1 was a gift from Dr. Lei Li. This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.

In this protocol we describe the method for analysis of DNA DSB repair with chromosomally integrated reporter constructs1,2 where DSBs are induced in vivo by transient expression a rare cutting endonuclease I-SceI3. The integrated assay provides the advantage of analyzing DSB repair within the chromosomal context. However, this protocol requires prolonged cell passaging, which may be problematic when working with primary cells.
An alternative approach is an extrachromosomal assay where reporter constructs are digested in vitro with I-SceI or HindIII endonucleases and then transfected into cells as linear DNA. The exctrachromosomal assay protocol4 is similar to the integrated assay described below with the following modifications. For the extrachromosomal assay omit the integration of reporter constructs and instead co-transfect the cells with 0.5 μg of NHEJ reporter plasmid linearized in vitro with I-SceI or HindIII and 0.1 μg of pDsRed2-N1 as transfection control. For the HR assay co-transfect 2 μg HR reporter plasmid linearized with I-SceI or HindIII and 0.1 μg pDsRed2-N1. Analyze the cells by FACS three days after transfection. The extrachromosomal assay allows for the analysis of DSB repair in primary isolates, avoiding extensive cell passaging, and facilitating the analysis of multiple cell lines.
1. Reporter Constructs for NHEJ and HR
The NHEJ reporter cassette5 contains a GFP gene with an engineered 3 kb intron from the Pem1 gene (GFP-Pem1). The Pem1 intron contains an adenoviral exon flanked by recognition sequences for HindIII and I-SceI endonucleases (Figure 1a) for induction of DSBs. The I-SceI sites are in inverted orientation. I-SceI has a nonpalindromic recognition sequence, hence two inverted sites generate incompatible DNA ends (Figure 1c). Incompatible DNA ends best mimic the naturally occurring DSBs. The intact NHEJ cassette is GFP negative as the adenoviral exon disrupts the GFP ORF. Upon induction of DSBs by I-SceI, the adenoviral exon is removed and NHEJ restores function of the GFP gene (Figure 2). The unique feature of this NHEJ reporter is that it can detect a wide spectrum of NHEJ events since the intron can tolerate deletions and insertions.
The HR reporter cassette1 (Figure 1b) is also based on the GFP-Pem1. In the HR cassette, the first exon of the GFP-Pem1 contains a 22 bp deletion combined with the insertion of three restriction sites, I-SceI/HindIII/I-SceI. The deletion ensures that GFP cannot be reconstituted by an NHEJ event. The two I-SceI sites are in inverted orientation, so that I-SceI digestion leaves incompatible ends (Figure 1c). The first copy of GFP-Pem1 is followed by a promoter-less/ATG-less first exon and intron of GFP-Pem1. The intact construct is GFP-negative. Upon induction of a DSB by I-SceI digestion the functional GFP gene is reconstituted by intramolecular or intermolecular gene conversion between the two mutated copies of the first GFP-Pem1 exon. Since the second copy of the GFP gene is lacking the first ATG codon and the second exon, crossing over or single-strand annealing will not restore the GFP activity. This design allows for the exclusive detection of gene conversion, which is the predominant HR pathway in mammalian cells (Figure 3).
2. Integration of the Reporter Constructs into the Genome
<ol>
 <li>Make a high quality preparation of NHEJ or HR reporter plasmid. For best results use Qiagen Endo Free Kit. Measure plasmid concentration and purity by absorbance at 260/280 nm.</li>
 <li>Linearize 10 μg of the reporter plasmid by digesting with 50 U of NheI in 50 μl (total volume of the reaction) for 6 hours in 37°C incubator (do not use the dry block, to prevent evaporation). Purify DNA from the digestion solution with Qiagen QiaexII Kit, elute DNA into 20 μl of 10 mM Tris-HCl pH 8.0. Measure DNA concentration and purity by absorbance at 260/280 nm. Usually, you will lose 20-30% of DNA during purification. Confirm the yield and digestion by running a small aliquot (2 μl) on a gel, along with an undigested reporter construct. The purified linearized reporter construct can be stored at 20°C.</li>
 <li>In preparation for transfection, bring the cells to the best growing condition. If starting from a frozen vial, split the cells twice before transfection. If starting from a plate of confluent cells, split them once.</li>
 <li>Grow the cells to 70-80% confluence (usually two days, if plated 5x105 cells/100 mm plate, for normal human fibroblasts). It is critical for the best transfection efficiency to bring the cells into logarithmic growth. Actively growing culture contains cells in M stage (seen as rounded cells attached to the surface). </li>
 <li>Transfect the cells using Amaxa Nucleofector following manufacturer's recommendations. For fibroblasts use NHDF solution and program U20. For transfection, harvest the cells from two 100 mm plates (~2x106 cells). It is critical not to over trypsinize the cells. Stop trypsinization as soon as 80% of cells detached from the plate. Resuspend the cells in NHDF solution. Cells are sensitive to NHDF solution, therefore, minimize the incubation time and mix the cells gently. Transfect the cells with 0.5 μg of linearized reporter construct. These transfection conditions, when used with normal human fibroblasts result in the integration of a single copy of a reporter construct for the majority of integrants. </li>
 <li>Select the cells with chromosomally integrated reporter constructs by adding 1 mg/mL geneticin (G418) 24h after transfection. Continue selection for 7-10 days.</li>
 <li>Pick the G418 resistant colonies (you can pick individual colonies or pool the resistant clones). Expand the culture to approximately five 100 mm plates. Freeze three plates for future use and expand remaining two plates to the desired cell number (usually ten 100 mm plates are sufficient for five transfections). </li>
 <li> When the cells reach 70-80% confluence (two days after plating 5x105 cells per 100 mm plate for fibroblasts), the cells are ready to be transfected with I-SceI expressing plasmid.</li>
</ol>
3. Induction of DSB by Transient Expression of I-SceI Endonuclease
<ol>
 <li>Transfect ~2x106 cells (two plates) of logarithmically growing culture with a mixture of 5 μg I-SceI expressing plasmid and 0.1 μg pDsRed2-N1 (Clontech) as a transfection efficiency control using Amaxa Nucleofector (use NHDF solution and U20 program for fibroblasts). </li>
 <li>At the same time prepare three calibration controls for FACS by trasfecting ~2x106 cells with (i) 5 μg EGFP-expressing plasmid; (ii) 5 μg pDsRed2-N1; (iii) 5 μg of a control plasmid that does not express a fluorescent protein (we are using a plasmid encoding HPRT minigene).</li>
 <li>Grow the cells for four days to provide sufficient time for expression of GFP and DsRed proteins.</li>
 <li> (Optional) Three days after transfection verify the efficiency of transfection and expression of GFP and DsRed proteins by fluorescent inverted microscope with filters for detection of GFP and DsRed.</li>
</ol>
4. Analysis of the GFP+ and DsRed+ Cells by Flow Cytometry
<ol>
 <li>Four days after transfection harvest I-SceI transfected cells and the control cells. Resuspend the cells in 500 μL of PBS and transfer cells to the FACS tubes. At this stage it is critical to harvest all the cells and have cells of round shape. To achieve this, it is recommended to use a longer trypsinization time or a stronger trypsin. Confirm that 99% of cells are detached from the plate and have round shape by observing the cells under microscope. Keep the cells on ice protected from light (cover ice bucket with foil). It is possible to store the cells on ice for a few hours. For the best results analyze the cells immediately after harvesting.</li>
 <li>Calibrate FACS with GFP, DsRed, and the negative controls. Adjust voltage and color compensation in order to include all the fluorescent cells in the analysis (Figure 4). Keep in mind that the fluorescent intensity of the experimental samples will be lower than that of the controls.</li>
 <li>Analyze I-SceI transfected cells by FACS. We count at least 20,000 cells for each treatment. To prevent potential interference between GFP and DsRed fluorescence it is recommended to keep the percent of GFP+ or DsRed+ cells below 25%. If the percentage of DsRed+ or GFP+ cells is higher reduce the amount of pDsRed2-N1 or I-SceI plasmid. In our experiments we only needed to adjust the amount of pDsRed2-N1 plasmid, but not of the I-SceI plasmid.</li>
 <li>The percent of GFP+ cells corresponds to the efficiency of DNA DSB repair and the percent of DsRed+ cells indicates the efficiency of transfection. Calculate the relative efficiency of DNA DSB repair as the ratio of GFP+ cells to DsRed+ cells.</li>
 <li> The repaired end junctions may be sequenced to test the accuracy of repair. The reporter constructs contain E.coli origin of replication and kanamycin resistance genes (Figure 1). This enables rescuing the constructs by digesting genomic DNA with EcoRI enzyme, recircularization, and transformation into competent E.coli cells. The rescued plasmids are then analyzed by restriction digestion and sequencing.</li>
</ol>
5. Representative Results
Typical FACS results of the control transfections and of NHEJ and HR experiments are shown in Figures 4 and 5. Fluorescent intensity and the number of fluorescent cells will be lower in the I-SceI transfected cells containing integrated NHEJ and HR constructs (Figure 5) compared to the controls (Figure 4) due to the difference in the copy number of GFP and DsRed constructs in these cells. Each cell in the experiment contains only one copy of the integrated reporter construct, thus successful repair events will reconstitute the GFP gene in a fraction of the cells. Transfection with 0.1 μg pDsRed2-N1 in human fibroblasts delivers approximately 1 plasmid copy per cell.
The efficiency of NHEJ and HR is calculated as a ratio of GFP+/DsRed+ cells. NHEJ is a more efficient process than HR in human cells2. In human fibroblasts the NHEJ efficiency is typically 0.6-1.3, and HR efficiency is 0.05-0.3. Since DSB efficiency is measured as a ratio GFP+/DsRed+ cells variations in the mixture of I-SceI and DsRed2-N1 plasmids may affect the result. The plasmid quality may also affect the results by changing transfection efficiency. Therefore, to obtain consistent measurements, it is important to use the same plasmid mix within one experiment. Furthermore, the DSB repair efficiency is affected by cell type, cell cycle phase, and chromosomal location of the integrated constructs.
 <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Nonhomologous end joining, homologous recombination diagrams; gene editing reporters, DNA sequence." data-altupdatedat="1788304762000" data-alt="Figure 1"> 
 Figure 1. Reporter constructs for the analysis of DNA DSB repair by NHEJ (A) and HR (B). (C) Incompatible DNA ends generated by digestion of two inverted I-SceI sites. SD, splice donor; SA, splice acceptor; shaded squares, polyadenylation sites; Neo/Kana, single ORF controlled by two promoters: SV40 conferring neomycin resistance in mammalian cells; and β-lactamase conferring kanamicyn resistance in E. coli; Ori, E. Coli pUC origin of replication.
<img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Non-homologous end joining diagram; DNA repair process; depicts GFP integration using enzymes." data-altupdatedat="1788304762000" data-alt="Figure 2"> Figure 2. Reporter construct for the analysis of NHEJ. In this construct the GFP gene is inactive; however upon induction of a DSB and successful NHEJ the construct becomes GFP+. Shown below is a repair product of this reporter by NHEJ.
<img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Homologous recombination diagram: GFP repair outcomes; GC, X-over, SSA, NHEJ processes depicted." data-altupdatedat="1788304762000" data-alt="Figure 3"> Figure 3. Reporter construct for the analysis of HR by gene conversion. Upon induction of the DSBs by I-SceI, HR by gene conversion (GC) reconstitutes an active GFP gene. Shown below are repair products of this reporter by the major DNA repair pathways: GC, crossing-over (X-
over), single strand annealing (SSA), and NHEJ. X-over repair product is shown for intramolecular recombination, exramolecular recombination will give the same product but located on one chromosome. Genes expressing active GFP protein (GFP+) are indicated by green color.
<img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Flow cytometry analysis, dot plots of fluorescent proteins GFP+ DsRed+ controls, fluorescence results." data-altupdatedat="1788304762000" data-alt="Figure 4"> Figure 4. Calibration of the parameters for FACS analysis. (A) Fibroblasts transfected with the pHPRT plasmid, used as a negative control for excluding auto fluorescent cells. (B) Fibroblasts transfected with pEGFP plasmid, used for setting the gating for GFP+ cells (green area). (C) Fibroblasts transfected with pDsRed2-N1 plasmid, used for setting the gating for DsRed+ cells (red area).
<img src="/files/ftp_upload/2002/2002fig5.jpg" alt="NHEJ and HR flow cytometry results; scatter plots of DsRed+ and GFP+ percentages in DNA repair." data-altupdatedat="1788304762000" data-alt="Figure 5"> Figure 5. Typical results of the analysis of NHEJ (A) and HR (B) in normal human fibroblasts with chromosomally integrated reporter constructs.

<ol><li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li>
<li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells%5BTitle%5D)+AND+%22DNA+Repair+%28Amst%29%22%5BJournal%5D)">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li>
<li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li>
<li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+repair+by+homologous+recombination%2C+but+not+by+nonhomologous+end+joining%2C+is+elevated+in+breast+cancer+cells%5BTitle%5D)+AND+%22Neoplasia%22%5BJournal%5D)">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li>
<li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li>
<li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li>
</ol>

No conflicts of interest declared.

The fluorescent NHEJ and HR reporter assays provide a quantitative way for separately measuring each DSB repair pathway in vivo. The assays are very sensitive, as FACS can reliably detect 10 GFP+ cells in 20,000 cells. The assays may be adapted for measuring repair events in "real time" by detecting the appearance of GFP+ cells within minutes or hours after induction of DSBs2. Furthermore, the analysis of GFP+ cells does not rely on additional cell proliferation, allowing DSB repair to be analyzed a...

<table><tbody><tr><td>EndoFree Plasmid Maxi kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Qiaex II Gel Extraction Kit</td><td>Qiagen</td><td>20021</td><td></td></tr><tr><td>Amaxa Nucleofector</td><td>Lonza Inc.</td><td>AAD-1001</td><td></td></tr><tr><td>Geneticin (G418)</td><td>Invitrogen</td><td>11811-031</td><td></td></tr><tr><td>pDsRed2-N1</td><td>Clontech Laboratories</td><td>632406</td><td></td></tr><tr><td>Round bottom tubes</td><td>BD Biosciences</td><td>352058</td><td>FACS tubes</td></tr></tbody></table>

analysis of dna double-strand break (dsb) repair in mammalian cells

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Demonstrated in this video protocol is a method for measuring the efficiency and accuracy of DNA double strand brake repair by NHEJ or HR in a mammalian cell line. The procedure starts with generating a cell line containing a chromosomally integrated single copy of NHEJ or HR reporter cassette. These reporter cells are then transfected with a mixture of I SCE one expressing plasmid and DS red and one plasmid.I sce. One endonuclease produces a double strand break in the reporter construct and DS RED serves as a transfection control. The percentage of GFP positive and DS red positive cells is then analyzed by fax in order to quantify the efficiency of NHEJ or HR if desired.The fidelity of repair is analyzed by rescuing the reporter products in e coli and sequencing the repair products. In this way, the efficiency and fidelity of doublet strand break or DSB repair in a given cell line is determined based on flow cytometric quantification of repair events and sequencing of the repair products. This method has many advantages over the existing methods for the analysis of double string brake repair.First of all, this method is specifically differentiates between a homologous recombination and non-homologous and joining. Then the method is highly quantitative, allowing the analysis of millions of cells by flow cytometry and then the method does not require extensive packaging of cells after repair is complete for selection of antibiotic resistant cls. And the lastly, the completion of repair can be monitored in real time by looking at appearance of green cells.And this method can help answer key questions in the a repair field such as measuring HR and an HEGA repair efficiency in virus mutant cell types or following drug treatments. Examine the A repair at different stages of cell cycle and measuring the rate of the repair So general. Generally individuals new to this method will struggle because it is important to achieve good transfection efficiency.In this procedure. Two reporter cassettes are used to assay for repair of double stranded DNA breaks. One cassette is used to examine non-homologous end joining or N-H-E-J-D-N-A repair.The other is used to look at homologous recombination or HR DNA repair the non-homologous end. Joining reporter cassette contains a GFP gene with an engineered three kilobase intron from the PEM one gene or GFP PEM one. In short, the P one intro contains an adenoviral exon flanked by recognition sequences for Hindi three and I SCE one endonuclease for induction of double stranded breaks.The I SCE one sites are an inverted orientation I SCE one has a non palindromic recognition sequence, hence two inverted sites generate incompatible. DNA ends incompatible. DNA ends best mimic the naturally occurring dss.An unarranged NAEJ cassette is GFP negative as the adenoviral exon disrupts the G-F-P-O-R-F. Upon induction of double stranded DNA breaks by ISEE one, the adenoviral exon is removed and NHEJ restores function of the GFP gene. The homologous recombination reporter cassette is also based on the GFP PEM one construct in the HR cassette.The first exon of the G FP PEM one contains a 22 base pair deletion combined with the insertion of three restriction sites. I SCE one Hindi three I SCE E one. The deletion ensures that GFP cannot be reconstituted by an NHEJ event here too.The I SCE one sites are in inverted orientation. So the I SCE one digestion leaves incompatible ends. The first copy of GFP PMM one is followed by a promoter less a TG, less first exon andt intro of GFP M1.The intact construct is GFP negative upon induction of a double strand break by I sce E one digestion.The functional GFP gene is reconstituted by a gene conversion event between the two mutated copies of the first GFP PMM one exon, other types of DSB repair, such as crossing over single stranded kneeling. And NHEJ will not reconstitute the GFP gene. Since the second copy of the GFP gene is lacking.The first A TG codon and the second exon resolution by crossing over or single-stranded kneeling does not restore the GFP activity. This design therefore allows for the exclusive detection of resolution by gene conversion, which is the predominant HR pathway in mammalian cells. To begin this procedure, use the endo free kit from Kyogen to prepare a high quality stock of NHEJ or HR reporter plasmid linearize 10 micrograms of the plasmid and purify the DNA from the digestion solution.Consult the accompanying written protocol for the details of plasmid preparation. In preparation for transfection. Make sure normal human fibroblasts are maintained under the best growing conditions.If starting from a frozen vial, split the cells twice before transfection. If starting from a plate of confluence cells split the cells once grow the cells to 70 to 80%confluence. This takes about two days for five times 10 to the fifth cells plated in a 100 millimeter plate.For best transfection efficiency, bring the cells into logarithmic growth. Actively growing culture contains cells in M stage seen as rounded cells attached to the surface for transfection harvest about two times 10 of the six cells from two 100 millimeter plates. It is critical not to over trypsin the cells stop trypsin as soon as 80%of cells have detached from the plate.Re suspend the cells in NHDF solution. Since cells are sensitive to NHDF solution, minimize the incubation time and mix the cells gently. Next, use the amaxa nucleo effector to transfect the cells with 0.5 micrograms of linearized reporter construct for normal human fibroblasts.These transfection conditions result in the integration of a single copy of a reporter construct for the majority of integr 24 hours after transfection at one milligram per milliliter of genetic or G four 18. In order to select the cells with chromosomally integrated reporter constructs, continue selection for seven to 10 days, then pick either individual G 4 1 8 resistant colonies or pool the resistant clones. Expand the culture to approximately five 100 millimeter plates.Freeze three plates for future use, and expand the remaining two plates to five times 10 to the fifth cells per 100 millimeter plate. Ten one hundred millimeter plates are usually sufficient for five transfect. Two days after plating when the cells containing the reporter construct reached 70 to 80%confluence with a mixture of five micrograms of I sce one expressing plasmid and 0.1 microgram of DS red plasmid.As a transfection efficiency control, use the AM maxon nucleo effector to transfect about two times 10 of the six cells from a logarithmically growing culture. Since DSP efficiency is measured as a ratio of GFP positive to DS red positive cells, variations in the mixture of ISCE one and DS red plasmid may affect the results. The plasmid quality may also affect the results by changing transfection efficiency.Therefore, to obtain consistent measurements, it is important to use the same plasmid mix within one experiment At the same time, prepare three calibration controls for facts for each control. Transfect about two times 10 of the six cells. The three controls are five micrograms of EGFP expressing plasmid, five micrograms of DS, red plasmid, and five micrograms.A control plasmid that does not express a fluorescent protein. Three days after transfection, verify the efficiency of transfection and expression of GFP and DS red proteins by fluorescent inverted microscope with filters for detection of GFP and DS.Red grow the cells for an additional day before fax analysis, four days after transfection harvest the I SCE one transfected cells and the control cells. At this stage, it is critical to harvest all the cells and have cells of round shape.To achieve this, use longer trypsin ization time or a higher concentration of trypsin. Examine the cells under the microscope to confirm that 99%of the cells are detached from the plate and have a round shape. Re suspend the cells in 500 microliters of PBS and transfer to fax tubes.Keep the cells on ice and protect them from light while it is possible to store the cells on ice for a few hours. For the best results, analyze the cells immediately after harvesting. Next, calibrate the facts with TFP DS RED and the negative controls.Adjust the voltage and color compensation in order to include all the fluorescent cells in the analysis. Keep in mind that the fluorescent intensity of the experimental samples is lower than that of the controls. After calibrating the facts with the controls, analyze the I SCE one transfected cells count at least 20, 000 cells for each treatment to prevent potential interference between GFP and ds.Red fluorescence keep the percent of GFP positive or DS red positive cells below 25%If the percentage is higher, reduce the amount of DS RED or I sce E one plasmid. The percent of GFP positive cells corresponds to the efficiency of D-N-A-D-S-P repair and the percent of DS red positive cells indicates the efficiency of transfection. Calculate the relative efficiency of D-N-A-D-S-P repair as a ratio of GFP positive cells to DS red positive cells.In order to determine the accuracy of the repaired injunctions, rescue the reporter constructs by digesting genomic DNA with eco R one enzyme circularization and transformation into competent e coli cells. This rescue is possible since the original constructs contain a bacterial origin of replication analyzed by restriction, digestion, and sequencing. These are typical facts, results of the control transections and of NHEJ and HR experiments.The fluorescent intensity and the number of fluorescent cells are lower in the I SCE one transfected cells compared to the controls. The difference in the fluorescence signal is due to the difference in the copy number of GFP and DS RED constructs in these cells. Each cell in the experiment contains one copy of the integrated reporter construct.Thus successful repair events reconstitute the GFP gene in a fraction of the cells. The efficiency of NHEJ and HR is calculated as a ratio of GFP positive to DS red positive cells. NHEA is a more efficient process than HR in human cells, in human fibroblasts, the NHEJ efficiency is typically 0.6 to 1.3 and HR efficiency is 0.05 to 0.3.This technique paved the way for researchers in the field of DNA repair to explore the roles of various factors in HEG and HR and study the kinetics and regulations of NHEG and HR in mammalian cells. After watching this video, you should have a good understanding of how to measure the efficiency and accuracy of HR and GJ in mammalian cells using a fluorescent assay.

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of γH2AX and 53BP1.

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Learn GFP-based fluorescence assays to quantify homologous recombination and nonhomologous end joining in mammalian cells. This method measures DNA double-strand break repair efficiency and accuracy.

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

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