Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

The overall goal of this procedure is to replace the bone marrow compartment of recipient mice with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein. In the absence of irradiation. This is accomplished by first conditioning the recipient mouse with serial intraperitoneal injections of the myelosuppressive compound busalan.

Next bone marrow cells are isolated from the femurs and tibia of a donor mouse ubiquitously expressing green fluorescent protein. Finally, donor bone marrow cells are transplanted into the conditioned recipient mouse via a tail vein injection. Ultimately, blood is collected from the transplanted mouse and analyzed by flow cytometry to estimate the extent of bone marrow chimes.

The main advantages of this technique over existing methods like total body irradiation, is that a high degree of bone marrow chimes can be established without the cytotoxic effects of total body irradiation and without the need for specialized facilities and equipment. Not only does this method allow insight into the accumulation of bone marrow derived cells into the central nervous system, but it can also be used to study such research topics as hematopoietic lineage development, immunobiology and leukemia. To condition recipient mice prepare diluted busulfan according to the text protocol and use IP injection to administer 20 milligrams per kilogram to recipient mice daily.

After euthanizing the mice according to institutional guidelines, under a laminar flow hood, spray a mouse with 70%ethanol. Lift the skin at the abdomen and with surgical scissors, make an incision through the skin from the abdominal cavity, up the leg towards the ankle holding the foot. Firmly pull the skin from the ankle towards the hip, exposing the leg tissue.

Trim away the muscle and fat from the femur to expose the hip joint while gently pulling on the foot. To extend the leg, press the scissors against the pelvic bones, cut just above the head of the femur, taking care not to cut the femur itself. To help maintain sterility, hold the leg by the foot and clean any remaining tissue from the femur using scissors and autoclave tissues to rub the bone surface.

Separate the femur and tibia by cutting through the knee joint and place the femur in a culture dish containing PBS incubate on ice. Next, remove and discard the fibula by cutting at the points where the fibula connects to the tibia. Then clean the tibia.

Remove the foot by cutting the tibia where the red marrow ends, and place the tibia in the culture dish with the femur and incubate on ice. Use forceps to hold the femur and with surgical scissors, carefully shave the distal ends off the bone. Remove as little of the bone as necessary to expose the medullary cavity with three milliliters of sterile PBS.

Fill a syringe and attach a 23 gauge needle. Carefully bore the needle into the medullary cavity and flush the bone marrow into a sterile culture dish. Use the needle point to scrape the medullary cavity to ensure removal of all desired cells following extraction.

Ensure that the red bone marrow is no longer visible and the bone now appears white. Use forceps to hold the tibia and with surgical scissors, carefully shave the end where the tibia was attached to the knee. To expose the medullary cavity, fill a syringe with three milliliters of sterile PBS and attach a 25 gauge needle.

Carefully bore the needle into the medullary cavity and flush the bone marrow into the dish of pooled bone marrow from the femurs. With a one milliliter pipette tip, gently tri the bone marrow to dissociate the cells. Pass the cell suspension through a 40 micron basket filter into a sterile 50 milliliter centrifuge tube.

Use PBS to rinse the dish to get any remaining cells and transfer the buffer through the filter into the centrifuge tube. Add PBS to the tube giving a final volume of 30 to 40 milliliters and centrifuge at 450 times G and four degrees Celsius for five minutes. Then remove and discard the supernatant.

Resuspend the pellet with three milliliters of erythrocyte lysing buffer incubate on ice for 8.5 minutes. Subsequently, add about 30 milliliters of sterile PBS to quench the lysing buffer centrifuge and discard the supernat. Use one milliliter of sterile PBS to re the pellet and keep on ice.

With PBS dilute a small amount of cell homogenate and use a hemo cytometer to count the cells. Dilute the cell suspension to eight times 10 to the six cells per milliliter and incubate on ice until injection. To perform the bone marrow transplantation, add 300 microliters of the diluted cell suspension to a 0.5 milliliter syringe.

For each mouse to be injected, be sure to remove any air bubbles. Place the mouse cage with the conditioned recipient mice on a heating pad and allow the lateral tail veins to dilate. Place a recipient mouse into an appropriately sized restraining device and wipe the tail with surgical gauze soaked with 70%ethanol firmly hold the tail and with the bevel up gently insert the needle into one of the lateral tail veins and inject the cell suspension.

Hold surgical gauze on the injection site until bleeding stops. Prior to introducing the mouse into a new clean cage, approximately two to three weeks after bone marrow transplantation, placed the mouse in a restraining tube and shave the fur on the posterior leg to expose the lateral saphenous vein. Next, apply a thin coat of petroleum jelly to the leg and use a 25 gauge needle to pierce the saphenous vein with a heparin coated capillary tube.

Collect about 50 microliters of blood, then use surgical gauze to stop the bleeding. Once bleeding stops, return the mouse back to the cage. Add the blood to a micro centrifuge tube containing one milliliter of fax buffer.

Mix the tube by inversion and store on ice centrifuge the blood samples at 900 times G for five minutes. After removing and discarding the supernatant, use 500 microliters of erythrocyte lysing buffer to resuspend the pellet and incubate on ice for 8.5 minutes. Subsequently, add one milliliter of fax buffer to quench the lysing buffer and centrifuge at 900 times G for five minutes.

After lysing and spinning a second time, according to the text protocol, check that the pellet is now white. If it is red, repeat the lysing Step a third time resus. Suspend the pellet in fax buffer and use flow cytometry to quantify the proportion of GFP positive cells.

This graph shows the levels of chimerism quantified weekly in the peripheral blood of mice conditioned with 100 milligrams per kilogram of busalan receiving a successful syngeneic bone marrow transplantation and mice conditioned with 80 milligrams per kilogram busalan receiving an unsuccessful allogeneic bone marrow transplantation. Climber levels greater than 80%are typically established by three to four weeks post bone marrow transplantation shown here are representative facts data of peripheral blood showing a successful and unsuccessful bone marrow transplantation three weeks after bone marrow transplantation. This flow cytometry data shows that a high degree of chimes remains established for at least one year in the bone marrow, and that conditioning with vehicle is not sufficient to induce bone marrow chimes.

While there are no significant differences in the levels of bone marrow chimes achieved using doses of 60 to 100 milligrams per kilogram busulfan GFP positive cells accumulate within the central nervous system in a dose dependent manner. This figure illustrates GFP positive bone marrow derived cell accumulation within the lumbar region of the spinal cord in a wild type mouse, and to a greater extent in a mouse model of the neurodegenerative disease am atrophic lateral sclerosis. When implementing this procedure, it's important to use syngeneic recipient and donor mice that are the same sex in order to minimize the possibility of transplant rejection Following this procedure.

Other methods like flow cytometry and immunohistochemistry can be utilized in order to study the mechanisms by which bone marrow derived cells enter the central nervous system.