The goal of this procedure is to generate retrogenic mice to determine the in vivo function of multiple t-cell receptors. This is accomplished by utilizing a retroviral construct that encodes the alpha and beta chains of the t-cell receptor, linked by a 2A peptide consensus motif. This method can actually help answer key questions in the immunology field, by screening multiple TCRs specific for multiple antigens, you can actually determine their in vivo function.
The main advantage of this technique is that once the retroviral construct is completed, the t-cell compartment of the retrogenic mice will be reconstituted approximately around seven weeks. Additionally, each retrogenic mouse is essentially a founder, so by analyzing multiple mice, the founder effect is eliminated. Demonstrating this part of the procedure will be Dr.Maria Bettini.
Set up for the bone marrow extraction by placing sterile dissection scissors and forceps in the surgical area and by chilling dissection medium in a 50 milliliter conical tube on ice. After euthanizing the mouse and confirming death by cervical dislocation, use dissection scissors to cut up and under the pelvic girdle to remove it. Then cut back down along the femur to the tibia and carefully remove the femur, tibia and humerus.
Transfer the bones into a dish of dissection medium in the tissue culture hood and use scissors and forceps to remove all surrounding tissue to obtain clean bones. Separate the bones by cutting at the joints, making sure to cut both ends off each bone. Then insert a 25 gauge needle attached to a 10 milliliter syringe containing dissection medium into a bone.
Apply pressure and flush all bone marrow through a 70 micron strainer, resting in a 50 milliliter conical tube. Periodically, push the bone marrow through the 70 micron filter using the plunger of a one milliliter syringe and rinse the strainer with medium. Centrifuge the bone marrow single cell suspension at 300 times g for 10 minutes at four degrees celsius.
Following the centrifugation, decant or aspirate the medium and resuspend each cell pellet in one milliliter of ammonium chloride based red blood cell lysis buffer. Incubate at room temperature for one minute. Next, quench the red cell lysis buffer by adding 10 milliliters of plating medium and then centrifuge as before.
After removing the supernatant, resuspend the bone marrow in five milliliters of plating medium and perform a cell count using neubauer counting chamber. The yield should be approximately five to 10 times 10 to the sixth cells per mouse, but this can vary between strains. Add 30 milliliters of plating medium, supplemented with freshly thawed IL-3, IL-6 and SCF to 150 millimeter tissue culture plates.
Then, add four times 10 to the seventh bone marrow cells to each plate and incubate over night at 37 degrees celsius with five percent carbon dioxide. Demonstrating this part of the procedure will be Thomas Lee. Next, plate three times 10 to the sixth retroviral producer cells in 150 millimeter plates containing 18 milliliters of plating medium.
Incubate the retroviral producer cells overnight. The next day, use a 10 milliliter syringe and 0.45 micron filter to harvest and filter the retroviral supernatant from the producer plates. Carefully replace the removed retroviral supernatant with 18 milliliters of fresh plating medium.
Supplement the filtered retroviral supernatant with IL-3, IL-6, MSCF and hexadimethrine bromide. Next, move on to harvest the bone marrow cells by first transferring the medium containing non-adherent cells into a sterile 50 milliliter tube. Then wash the plate vigorously with 10 milliliters of PBS and add this to the 50 milliliter conical tube.
After centrifuging the cells at 300 times g for 10 minutes at four degrees celsius, decant the supernatant and then resuspend the cells in one to three milliliters of plating medium. After performing a cell count, place bone marrow cells into the soup. Plate this cell mixture at a volume of three milliliters per well in a six well tissue culture plate.
Wrap the plates containing bone marrow cells in plastic wrap to minimize gas exchange and then spin the plates at 1, 000 times g for 60 minutes at 37 degrees celsius. After the spin in complete, remove the plastic wrap and place the plates in a 37 degree celsius carbon dioxide incubator for 24 hours. After 24 hours, collect fresh viral supernatant from the viral producer cell line.
At this point, the cells should be well spaced with a defiant morphology. Next, carefully aspirate the top two milliliters of medium from each well of the six well plate containing the bone marrow cells. Avoid aspirating the bone marrow cells that are usually concentrated in the middle of the well.
Add three milliliters of freshly supplemented viral supernatant to each well of the six well plate containing bone marrow cells. After wrapping the plates in plastic, as before, repeat the spin transduction, and then place the unwrapped plates in the tissue culture incubator. After 24 hours, on day three, remove and replace two milliliters of media from the wells.
On day four, collect the bone marrow cells from the six well plate by vigorously washing with PBS and then collecting the cell suspension in a 50 milliliter tube. After centrifuging the cells at 300 times g for 10 minutes at four degrees celsius, decant the supernatant and resuspend the bone marrow in one milliliter of PBS plus 0.5 percent heat inactivated FBS. Place the tube containing the cells on ice.
Then, after performing a cell count, resuspend the bone marrow in a sufficient volume of PBS plus 0.5 percent heat inactivated FBS, to inject a minimum of two times 10 to the sixth cells in a 200 to 300 microliter volume per mouse. Finally, with a micropipetter, take a 10 microliter sample of each bone marrow condition and add this to 100 microliters of PBS for analysis of transduction efficiency by flow cytometry. Transduction efficiency of bone marrow cells was analyzed based on the fluorescent marker ametrine.
The left panel shows the forward and side scatter of bone marrow cells after five days in culture. The right panel is gated as ametry impositive and includes the transduced bone marrow cells. A successful bone marrow transduction will yield a percentage of fluorescent positive cells between 25 and 70 percent.
This image shows t-cell receptor retrogenic t-cells in peripheral blood at six weeks post bone marrow reconstitution. After lysis of red blood cells, the remaining cells were stained for t-cell receptor beta. The right panel shows a representative analysis for ametrine expression and anti t-cell receptor beta.
After watching this video, you should have a good understanding of to retrovirally transduce and culture bone marrow cells to regenerate retrogenic mice.
