The overall goal of the following experiment is to test for specific variants in the BRCA one protein using an in vitro homologous recombination assay. This is achieved by replacing the endogenous wild type BRCA one protein with the variant being tested through the transfection of both short interfering RNA, specific to BRCA one and plasmid for re expressing test BRCA one variant proteins into cells as a second step, the short interfering RNA and the plasmid for expressing mutant BRCA one is RET resected in addition to a plasmid for expressing the ISCE one protein, which causes DNA damage at a specific site in the DNA genome of the test cell. Next, the cells are incubated for two to three days in order to allow for the DNA damage and repair to occur results are obtained that show whether specific protein variants of the BRCA one protein function in DNA repair by homologous recombination based on flow cytometry analysis of GFP expressing cells.
This technique combines two established methods in order to create a versatile system for analyzing specific protein variants in the repair of DNA damage by homologous recombination. The main advantage of this technique over existing methods is that previously established methods relied on cell lines that had a genetic knockout of BRCA one. We use a more manageable cell line derived from HELOC cells that enables BRC one depletion by RNA interference of the endogenous protein an add back of test protein in these cells.
Now expressing a variant BRC one protein we can test for homologous recombination. Generally, individuals new to this method will struggle because of excessive cell death during the procedure. It's important to have a transection condition carefully set maximizing the effect of recombination reaction while limiting cytotoxicity.
Prior to the start of this protocol, the hela DR 13 nine cell line was established by transecting hela cells with P-D-R-G-F-P Maintain the cell line on 10 centimeter Petri dishes in pur mycin for selection of stably transfected cells. The recombination substrate in the P-D-R-G-F-P plasmid and in the stable transformants contains two inactive alleles of GFP. One allele is inactive due to the inclusion of the 18 base pair restriction enzyme recognition site for ISCE one.
In its coding sequence expression of ISCE one in these cells creates a double stranded DNA break or DSB on the second allele of inactive GFP. The portion of its sequence correlated with the I SCE E one site on the first allele is wild type. This second GFP allele can function as a sequence donor for homologous recombination repair of the DSP and would result in an active GFP allele, which is readily detected by flow cytometry the day before the first transfection.
Obtain a 10 centimeter dish of hela DR 13 nine cells that are at least 90%confluent trypsin eyes the cells in one milliliter for two to three minutes. Stop the reaction by adding nine milliliters of D-M-E-M-F-B-S and mix the cell suspension well by pipetting. Add 40 microliters of tryps inized cells per well of a 24 well plate in 0.5 milliliters of D-M-E-M-F-B-S incubate the cells at 37 degrees Celsius and 5%carbon dioxide overnight.
On day one, cells should be between 40 and 50%confluent. Begin transfection by mixing both the irna to deplete endogenous b RCA one and the B RCA one mutant expressing plasmid in optimum media. Then combine the nucleic acids with lip in optimum media.
Proceed with the lipectomy transfection as per the manufacturer's instructions following transfection. Return the cells to incubate. Replace the media four to six hours later with medium that does not contain antibiotics.
Return the cells to incubate overnight on the second day. Trypsin is the cells by adding 0.3 milliliters of trip le trypsin reagent to each well after a brief incubation at 37 degrees Celsius, loosen the cells by pipetting the cells up and down in the trypsin reagent. Once trypsin is transfer the cells from the 24 well plate to a six well plate containing two milliliters of D-M-E-M-F-B-S to halt tripsin activity.
Allow the six well plate to incubate overnight under the same conditions as before two days from the initial transfection. Prepare the SIR A, the BRCA one mutant expressing plasma DNA and the ISCE one expressing plasmid in 62.5 microliters of Optum. Mix this solution with 2.5 microliters of LIPECTOMY 2060 2.5 microliters of Optum.
Proceed to transfect the cells after a four to six hour incubation. Replace the media with D-M-E-M-F-P-S lacking antibiotics. Return the cells to the incubator on day six.
Trypsin is the cells in each well by adding 0.5 milliliters of trip e trypsin reagent. Stop the reaction by adding two milliliters of D-M-E-M-F-B-S collect the cells by centrifugation at 1000 RPM for five minutes at room temperature. Resuspend the cells in one milliliter of fresh D-M-E-M-F-B-S analyze 10, 000 cells per well for GFP expression.
By flow cytometry, single live cells are gated using forward and size scattered parameters. GFP positive cells are directed using the GFP fluorescence detector and results are presented as a histogram. Remaining cells can be replated for photography by fluorescence.
Microscopy of desired BRCA one regulates the pathway for repair of double stranded DNA breaks by homologous recombination. Those cells which have undergone repair by homologous recombination are readily detected by fluorescent microscopy. Depletion of BRCA one by RNAi results in a sharp decrease in the detected GFP positive cells.
The output from the FCA is a histogram with GFP intensity per cell on the x axis and the number of cells on the Y axis. In the absence of transfected, I SCE one expressing plasmid. There are no GFP positive cells detected.
The results in columns two through six were all taken from cells in which ISC one expressing plasmid was transfected on day three. In a typical set of results from a single experiment, 15.5%of the cells are GFP positive after I SCE one expression and control RNAi depletion for an irrelevant gene by comparison depletion of BRCA one and add back of an empty plasmid vector. Reduce GFP conversion to 0.7%Addition of exogenous wildtype BRCA one fully restored homologous recombination as detected by obtaining 15 to 16%GFP positive cells.
Similarly, expression of another variant of unknown significance. The BRCA one I 21 B mutation fully restored. BRCA one dependent homologous recombination function add back of the BRCA one M 18 T mutant had a similar effect as vector control.
Since this variant expressed at similar levels as the endogenous protein, it suggests that the BRCA one M 18 T variant is non-functional in homologous recombination. While attempting this procedure, it is important to remember to monitor the growth of the cells, that the cells are growing slowly and at low conf fluency and the amount of transfection mixture used must be reduced. It is also important to remove the transfection medium in a timely fashion.
We find the days following transfection, there's a high amount of cytotoxicity if antibiotics are included in the medium. For this reason, all the medium after day one lack antibiotics After its development. This technique paved the way for researchers in the field of breast cancer research to explore the genetics of BRC one variants with unknown clinical significance in cancer clinics.
We hope that the functional BRC one assay developed in this work will enable improved prediction of the effects of BRC one variants on cancer predisposition.
