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.
