The overall goal of the following experiment is to alter the expression of a gene and then to measure its impact on somatic hypermutation. This is achieved by transecting Bosque 23 cells to produce viral particles containing either an overexpression or an SHRA containing construct. As a second step, ramo cells are infected with the viruses resulting in either knockdown or overexpression of the gene of interest.
Next single cell clones are grown for several weeks in order to allow the cells to accumulate mutations. Finally, the effect of the altered gene expression can be determined based on analysis of IgM loss and by sequencing of the IG genes. This method could help identify novel factors involved in antibody diversification.
Begin by elo, quoting 600 microliters of warm mented DMEM into a 1.5 milliliter micro centrifuge tube. Then being careful to pipette directly into the media to L 30 microliters of FU gene six into the tube vortex the solution for less than a second, and then incubate the tube at room temperature for five minutes. Next, for lentiviral infection such as this one, add six micrograms of S-H-R-N-A construct and 1.5 micrograms each of P-V-S-V-G-P-M-D-L-G-P-R-R-E and PRSV rev to the DMEM FU gene six mix.
Vortex the solution briefly and then incubate the two bit room temperature again, this time for 25 minutes. Finally, transfect a Bosque 23 cell culture at 50 to 60%co fluency with the D-N-A-D-M-E-M FU gene six. Mix and incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.
The next day, add five milliliters of DMEM to the cells and return them to the incubator for another 24 hours. On the third day after transfection, use a 10 milliliter syringe to harvest 10 milliliters of media from the transfected box 23 cells, and then attach a 0.45 micrometer filter to the end of the syringe. Collect the filtered media into a clean 15 milliliter polypropylene tube 24 hours before infection seed two milliliters of two times 10 of the fifth cells per milliliter ramo cells in complete RPMI into a six well plate the following day, add three microliters of a 10 microgram per milliliter poly brain solution, and one milliliter of virus to the ramo cells, and mix the cells suspension well by pipetting.
For the uninfected control, add one milliliter of complete RPMI and three microliters of poly brain to one of the wells instead of virus. Also infect one well with a matched negative control and another with a GFP containing vector for determining infection efficiency by fax. Now centrifuge the six well plate at 3000 GS for 90 minutes at room temperature, and then return the plate to the incubator for 48 hours.
Finally, spin down the infected cells in a 15 milliliter polypropylene tube at 400 Gs for four minutes. At room temperature, then aspirate the media being sure to not disturb the cell palate and resuspend the cells in fresh RPMI. After counting the cells, ute an eloqua of the infected ramo cells to 10 cells per milliliter.
In a solution of RPMI conditioned media and FBS to a final volume of 10 milliliters for each seeded plate. Then use a multi-channel pipette to seed. 96 well plates with 100 microliters per well of the diluted cells.
After incubating the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide, use a microscope to check each individual well. To confirm the presence of cells as the proper depth for identifying single cells can be tricky to determine. First, try focusing on the well numbers imprinted at the bottom of the 96 well plate.
Then try searching for a cell after two weeks. Check for cell colonies along the edges of the wells using permanent ink. Mark the wells with robust cell growth, and then transfer the colonies to 24 well plates in one milliliter of RPMI for further incubation.
After several days of growth, transfer the cells to a six well plate and continue incubating the cultures. Three weeks after seeding the cell colonies in the six well plate Eloqua, five times 10 to the fifth cells from each well into a 1.5 milliliter micro centrifuge tube. At this time, also harvest the cells from each of the control and HRNA knockdown wells, as well as one sample for an unstained control.
Now spin down all the cells at 400 Gs for four minutes at room temperature, twice washing the cells with one milliliter of PBS for the second spin. After the second wash, we suspend the unstained control pellet in 50 microliters of PBS and the other pellets in PBS plus, a one to 20 dilution of PE labeled anti-human IGM antibody. Then incubate all the cell samples on ice for one hour after washing the cells two more times with PBS as before, discard the snat and resuspend all the pellets in 100 microliters of PBS.
Next, transfer the resuspended stained and control unstained cells to fax tubes and then analyze the cells by flow cytometry. Compare the percentage of IgM negative cells from the controls versus the S-H-R-N-A knockdown cells. A retroviral overexpression vector for a ID was transfected into bosque 23 cells.
Viruses were then harvested and used to infect Ramos cells, both wild type or wt and a ID overexpressing or a ID.High cells were single cell seated and grown for three weeks, at which point they were analyzed for gene expression by QR TPCR as expected, the QR Tpcr R results show a marked increase in a ID expression following infection of cells with an A ID overexpression vector. IGM loss on the infected cells was evaluated by facts as well. Surface IGM was measured by facts from the previous figure at the three week time point, and the percentage of IGM loss was calculated for wild type and a ID high cells and represented graphically here as expected a ID over expressing cells exhibited a greater loss of surface IGM.
In a separate experiment, lentiviruses containing either an irrelevant S-H-R-N-A as a control or an S-H-R-N-A against a high fidelity DNA repair factor expected to protect against SHM were made in bosque 23 cells. The lentiviruses were then infected into an a ID high clone of Ramos cells. Again, gene expression was analyzed and again, as expected levels of the DNA repair factor were observed to drop in the presence of a targeted S-H-R-N-A against that factor i GM loss in the single cell clones was analyzed after three weeks as well, since the repair factor is thought to provide protection against mutations during SHM, an increase in IGM loss and SHM in the absence of the factor occurs as can be observed in this figure.
After watching this video, you should have a good understanding of how to perform viral infections of OVEREXPRESSION or S-H-R-N-A containing constructs in the raw B cell line, and then how to measure somatic hypermutation in these cells.
