This method can help answer key questions in cancer therapeutics fields, such as identification of key genes involved in oncogene dependence. The main advantage of this technique is that it utilizes the oncogene dependence that is common to many kinase driven cancers therefore it is applicable to multiple tumor types. Though this method can provide insight into leukemia, it can also be applied to other systems, like solid tumors such as lung cancer, brain tumors, and pancreatic tumors.
Generally, individuals new to this method will struggle due to lack of experience in molecular biology and cellular physiology. To begin, harvest the leg bones from a euthanized mouse. Clean the tissues from the bones with a scalpel.
Then place the bones in cold PBS on ice and crush the bones with a pestle. After this, filter the cell suspension through a 70 micrometer cell strainer into a 50 milliliter screw cap tube with a 10 milliliter pipette. Wash the mortar and pestle multiple times with cold PBS and add the cell suspension to the 50 milliliter tube.
Next centrifuge the bone marrow and remove the supernatant with vacuum and glass pipette. Resuspend the cell pellet in 5 milliliters of PBS. Using a pipette, slowly add the suspended bone marrow to the top of room temperature poly-sucrose.
After this, spin the poly-sucrose at 400 Gs for 20 minutes with the brake turned off. After centrifugation, collect the cloudy total mono-nuclear cell interface with a pipette and transfer it to a new 15 milliliter tube. Bring the volume to 10 milliliters with PBS and remove 20 microliters for counting.
After centrifugation, discard the supernatant and place the pellet on ice. Next, resuspend the cell pellet in PBS with EDTA and BSA. Add CD117 microbeads to the cell suspension, and vortex to mix.
After this, incubate the suspension for 10 minutes at 4 degrees Celsius. Then bring the volume up to 10 milliliters with more PBS with EDTA and BSA and centrifuge at 1200 G's at 4 degrees Celsius for 5 minutes. Use a vacuum to remove the supernatant and suspend the cell pellet in PBS with EDTA and BSA.
Then add 3 milliliters of PBS with EDTA and BSA to a magnet stand with a magnetic column and allow it to flow into a collection tube. After the buffer finishes flowing through the column, add the cell suspension and allow it to flow through the column into the collection tube. Next add 5 more milliliters of PBS with EDTA and BSA to the column and flush it immediately with the plunger.
Remove the plunger and repeat the flush before counting the cells. After centrifuging the cells, remove the supernatant and suspend the cell pellet in complete IMDM for culture. Culture the cells overnight at 37 degrees Celsius with 5%carbon dioxide.
First, combine the retroviral plasmid DNA with the packaging plasmid, calcium chloride and water in a 1.5 milliliter tube. Then add 500 microliters of HBS to another 1.5 milliliter tube. Add the transfection mixture drop-wise to the tube containing HBS while simultaneously mixing with the vortexer set to the lowest setting.
Incubate the transfection mixture at room temperature for 20 to 30 minutes. During the incubation period, add chloroquine to the HEK cells and keep them in the incubator. After adding the transfection mix, incubate the cells for 8 hours in a 37 degrees Celsius 5%CO2 incubator changing the cell media by adding 14 milliliters of fresh DMEM10 media very slowly.
Pipetting slowly against the wall of the dish helps to prevent any stripping of HEK cells. Then incubate the cells overnight in the 37 degrees Celsius 5%CO2 incubator. Use a 10 milliliter syringe to collect the viral supernatant.
Then attach a 0.45 micrometer syringe filter to the syringe. Plunge the viral supernatant into a new collection tube. Collect the viral supernatant approximately 16 hours later.
After this, add two milliliters of recombinant human fibronectin fragment in PBS to untreated 6 well culture plates. The next day, use a pipette to remove the fibronectin from the plates. Block the plates with 2%BSA in PBS and incubate the plates at room temperature for 30 minutes.
Use a vacuum to remove BSA and wash the plates with PBS before removing it with a vacuum. After this, immediately add the filtered viral supernatant and centrifuge the plate for two hours. Use a vacuum to remove the supernatant and spin the plate again.
Next remove the supernatant with a vacuum and wash the viral coated wells with PBS. Remove the PBS with a vacuum, add the previously cultured c-Kip plus mouse cells to the viral coated wells. After centrifugation, incubate the cells at 37 degrees Celsius and 5%carbon dioxide.
48 hours after transduction, pellet the cells via centrifugation and resuspend them in FACS buffer 1. First thaw the methyl cellulose with cytokines for mice at room temperature. Then aliquot 3 milliliters of methyl cellulose in a FACS tube.
Add 100 microliters of the cell suspension with the appropriate inhibitors to 3 milliliters of methyl cellulose. Then vortex the suspension on high for 10 to 30 seconds. After most of the air bubbles escape, use a 1 milliliter syringe to plate 1 milliliter of the media in three replicate plates.
Place the plates in a large Petri dish along with one open dish containing sterile water. Then cover the large Petri dish and incubate the cells at 37 degrees Celsius. Prepare iodonitrotetrazolium chloride stain by dissolving 10 milligrams of the iodonitrotetrazolium chloride in 10 milliliters of water.
Filter sterilize the staining solution with a Luer lock syringe equipped with a 0.2 micrometer filter. In the tissue culture hood, add 100 microliters of the staining solution drop-wise around the CFU plate. Then incubate the plates overnight at 37 degrees Celsius in a cell culture incubator.
Finally, after the colonies have turned a dark red brown color, use a white background in the sterile tissue culture hood to take photos of the stained CFU plate. In this protocol, multiple unbiased gene expression analyses were used to identify the genetic component that orchestrates oncogene addiction. To validate the role of c-Fos and and Dusp1 as the therapeutic target in leukemia, their overexpression was confirmed with cells expressing the BCR-ABL1 oncogene.
Analysis via RT-qPCR and western blotting confirmed that both were induced by BCR-ABL1 and that their expression is augmented by growth factor IL-3. YFP plus expressing cells were sorted via FACS for further in vitro and in vivo assays. The results demonstrated that deletion of c-Fos and Dusp1 alone inhibited CFU numbers.
Cells lacking both c-Fos and Dusp1 were significantly compromised in their ability to form colonies and Imatinib treatment wiped out all of the CFUs indicating that the loss of c-Fos and Dusp1 is synthetically lethal to BCR-ABL1 expression. After this development, this technique paved the way for researchers in the field of cancer to explore the genetic networks and genes driving oncogene dependence and how these genes affect their particular response in mouse models and human patients. Don't forget that working with retroviruses and patient samples can be extremely hazardous and safe laboratory practices should be observed while performing this procedure.
