The overall goal of this procedure is to generate mice expressing the gene of interest in hematopoietic cells using the transduction-transplantation method. Within a short period of time, this method can help answer whether your gene of interest induces a hematologic malignancy. The implications of this technique extend towards therapy, or diagnoses of hematological malignancies, because of the expedited development of mouse models that can test putative therapeutics.
Although this method can provide insight into hematological malignancy, it can also be used to study the role of a gene of interest of normal hematopoiesis. Generally, individuals new to this experiment will struggle, as it requires multiple steps. Also, generation of a high titer virus is critical to the success of this experiment.
Visual demonstration of this procedure is important, as working with mice can be difficult, and requires practice. After anesthetizing a mouse, and checking for depth of anesthesia according to the text protocol, position the mouse on its side, and using the thumb and index finger, restrain the animal so that the eye protrudes slightly from the head. To inject 150 milligrams per kilogram of 5-flurouracil, or 5-FU, via a retro-orbital injection, insert a 27-gauge half-inch needle at a 45-degree angle with the bevel facing up in a medial direction, and stop when the needle is halfway inserted.
Slowly inject the solution, and then carefully remove the needle. Examine the mouse for any signs of injury, such as swelling or bleeding, and ensure that the mouse recovers and begins to walk. Five days later, after sacrificing the animal according to the text protocol, use 70%ethanol to sterilize the mouse by spraying generously until the fur becomes wet.
With dissecting scissors, make an incision in the skin, and dissect the fascia from the leg muscles. Then, dissect the leg away from the mouse by severing the femur at the hip, and the tibia at the ankle. Bend the leg into an L-shape and use dissecting scissors to separate each leg bone by cutting at the knee joint.
Then, gently scrape the muscle towards one end of the leg bone until the cartilage is disengaged. Next, using a syringe with a 27-gauge half-inch needle, in PBS supplemented with 2%FBS, flush each leg bone over a 100 micrometer nylon membrane into a 50 milliliter conical tube, until the bone turns white to maximize the number of cells harvested. With the plunger of a syringe, mash the cells through the nylon membrane into the 50 milliliter conical tube to obtain a single cell suspension.
Repeat with the other mouse leg. Centrifuge the harvested cells at 400 times g in four degrees Celsius for 10 minutes, and discard the supernatant. Then, at five milliliters of ammonium chloride potassium, or ACK, to re-suspend the bone marrow.
Incubate the cell suspension on ice for 10 minutes to lyse the red blood cells. To stop the lysis, use PBS to fill the tube. Then, centrifuge the cells again, and discard the supernatant.
With five milliliters of PBS, re-suspend the cells, and by Trypan blue exclusion, use a hemocytometer to count the cells. Now, add 2x pre-stem cocktail to re-suspend the cells at two times 10 to the sixth cells per milliliter. Then, plate two milliliters in the wells of six-well plates.
Incubate the cells at 37 degrees Celsius, and 5%carbon dioxide overnight. After the incubation, add two milliliters of viral supernatant to each well, followed by eight micrograms per milliliter of polybrene. Spin the plates at 1500 times g in 30 degrees Celsius for 90 minutes.
Then return the plates to the incubator overnight. Pipette the cell suspension into conical tubes, and centrifuge at 400 times g in four degrees Celsius for 10 minutes. Use 2x pre-stem cocktail per well to re-suspend the cells, and re-plate in the original wells.
After preparing recipient mice for transplantation according to the text protocol, harvest transduced donor cells from the six-well plates by gently pipetting supernatant into conical tubes. Use one milliliter of PBS to wash each well, and collect the residual cells in the tubes. Add 0.5 milliliters of 0.05%trypsin to the wells, and incubate at 37 degrees Celsius for five minutes.
Next, add one milliliter of D-10 to the wells, and detach any remaining cells by gently pipetting. Then, add the cells to the respective tubes and pellet at 400 times g in four degrees Celsius for 10 minutes. With PBS, wash the cells again.
Then, after spinning, use five milliliters of PBS to re-suspend the cells, and count them using Trypan blue exclusion and a hemocytometer. For injection into mice, re-suspend five times 10 to the fifth, through two times 10 to the sixth cells in 50 to 100 microliters of PBS. Position the mouse on its side, and restrain it by using the thumb and index finger, so that the eye protrudes slightly from the head.
With the bevel facing up, insert the needle lateral to the medial canthus at a 45-degree angle, and inject. Following the injection, examine the mouse for any signs of injury, such a swelling or bleeding, at the injection site, and refer to the text protocol for additional post-injection care. Finally, analyze GFP expression in blood, according to the text protocol.
This figure illustrates how to calculate relative viral titer where the percent GFP is calculated from events collected from the live cell gate. Shown here is a titer with more than one viral particle per cell, and these samples represent titers with single viral particles per cell. Ideally, at least 50%of GFP-positive cells would be detected with a one to 30 dilution of viral supernatant.
In this experiment, a successful transplant recipient is presented where CALRdel52 mice exhibit an MPN phenotype with increased platelets, increased megakaryocytes in the bone marrow, and bone marrow fibrosis. Once mastered, this technique can be done in eight days, if performed properly. While attempting this procedure, it is important to follow every step of the protocol.
After its development, this technique paved the way for researcher to explore the effects of specific genes in hematopoiesis. After watching this video, you should have a good understanding of how to produce mice expressing your gene of interest in hematopoietic cells.
