The overall goal of this procedure is to stably heritability express a gene of interest. In this case the class switch regulator A ID within primary B lymphocytes from mice deficient in this gene, splenic B cells are harvested, purified, and activated using anti CD 40 and IL four A construct encoding A ID is transfected into a packaging cell line to produce viral particles containing the gene. Next, the pre activated B cells are infected with the recombinant retrovirus produced by the packaging cells.
The cells are then cultured for a further 47 to 72 hours to allow for growth, proliferation and expression of the introduced construct. Finally, the cells are stained and analyzed by flow cytometry for the presence of class switch surface antibodies. The main advantage of this technique over existing methods like mouse modeling, is that it requires a great deal less financial and technical resources, while still providing an efficient system of structure and function analysis of a gene of interest Using careful sterile technique.
Harvest the spleens of a ID deficient mice age three to six months, store the organs in cold PBS containing 15%FBS on ice until all the spleens have been harvested. Homogenize the spleen by forcing the tissue through a mesh screen placed over a six well plate containing five milliliters of PBS supplemented with 2.5%FBS transfer the homogenate to a 15 milliliter falcon tube centrifuge the homogenized tissue at four degrees Celsius and 1000 RPM for 10 minutes. Decant the supernatant and briefly flick the tube to loosen the cell.
Pellet resuspend the cells in 10 milliliters of red blood cell lysis, buffer and incubate the mixture at room temperature for seven minutes. To avoid contamination by red blood cells, centrifuge the lysed cells to wash them and decant the supernatant Resus. Suspend the pellet in 10 milliliters of PBS containing 2.5%FBS count the cells at a one to 1000 dilution using a standard hemo cytometer using trian blue to exclude any dead cells.
Approximately 20 to 30 million cells can be obtained per spleen, centrifuge the cells once more and resuspend them in 0.5%B-S-A-P-B-S containing two millimolar EDTA at a concentration of 10 million cells per 90 microliters. Add anti CD 43 antibody coated magnetic beads to the cells to specifically bind non B cells. Use 10 microliters per 10 million cells.
Mix well and incubate the cells for labeling at four degrees Celsius for 20 minutes. Once the cells have been labeled, wash them by topping up the tube with 2.5%F-B-S-P-B-S and centrifuging the sample. After having removed the supernatant Resus, suspend the cells at a concentration of 100 million per milliliter in 0.5%B-S-A-P-B-S containing EDTA mount a magnetic sorting column on a magnetic separator.
Position a conical tube directly underneath the column to collect the flow through and load the column with three milliliters of cold 0.5%B-S-A-P-B-S to prime and wash. Once the wash fluid has flowed through, load the sample in the column, load only one milliliter of cells per column and use multiple columns if necessary. Allow the cells to passively flow through the column and collect the unbound cells in the conical tube under the column.
Wash the column four times with three milliliters of 0.5%B-S-A-P-B-S while continuing to collect the flow through. Once the column has been washed, it can be discarded. Keep the flow through in which CD 43 negative resting B cells will be enriched.
Centrifuge the flow through and resuspend the pellet in 10 milliliters of complete RPMI. Medium count the cells and adjust the volume of medium to obtain a concentration of 1 million cells per milliliter. In order to stimulate B-cell growth and proliferation.
Supplement the medium with recombinant IL four and anti CD 40 antibody such that the final concentrations are 20 micrograms per milliliter and one microgram per milliliter respectively and transfer the cells to a culture flask. Inq incubate the culture for 72 hours, splitting the cells daily with IL four and anti CD 40, supplemented medium to maintain the cell concentration at 1 million per milliliter. Use 60 to 70%confluent phoenix cells as producer cells to be used for transfection at this confluence cell count should be 5 million cells per 10 centimeter plate one hour prior to transfection.
Replace the media with fresh complete media for Phoenix cell growth in a sterile einor tube. Add 100 micrograms of packaging construct DNA 250 microliters of 0.5 molar calcium chloride and adjust the final volume to 500 microliters with sterile water. Using a one milliliter pipette vigorously.
Mix this solution with 500 microliters of two XHNP in a 15 milliliter polypropylene tube. Allow this mixture to rest at room temperature for eight to 10 minutes. During this time, a precipitate will form, add the transfection mixture dropwise to the cells while swirling the medium to evenly distribute the mixture throughout.
Incubate the cells at 37 degrees Celsius for 12 hours, 24 hours. Post transfection. Replace the medium with eight milliliters of complete B-cell growth medium.
Then return the cells to the 37 degrees Celsius incubator, 48 hours post transfection, harvest the supernatant, which should now contain virus. Pass the supernatant through a 0.45 micron filter to remove any cell debris. Mix three milliliters of the virus containing supernatant with three milliliters of pre activated B cells from the previously described culture containing 1 million cells per milliliter in order to enhance viral absorption to the cells.
Add poly brain at a final concentration of 16 micrograms per milliliter. Infect the cells by centrifuging the mixture at 2, 500 RPM for 90 minutes at 30 degrees Celsius immediately after the spin. Incubate for a further 48 to 72 hours at 37 degrees Celsius to allow for cell growth and proliferation.
Using a flow cytometer analyze the cells for surface expression of B two 20 and surface IgG one. The presence of IgG one at the surface of the cells indicates that class switch recombination has occurred as a result of successful rescue via the retroviral complementation of the a ID gene. This graph shows the detection of IgG one at the surface of B cells with a ID, but not the empty construct indicating that class switch recombination has occurred as a result of a ID rescue.
Don't forget that working with recombinant retrovirus can be potentially hazardous and precautions such as appropriate institutional guidelines should always be taken while performing this procedure.
