We are here at the Molecular Oncology Research Institute at Tufts New England Medical Center. My name is Christina Lesco and I'm a postdoctoral fellow in Dr.Rick Van's lab. Our lab works on studying human immunogen leukemia, CML and CML like diseases.
We are modeling this human disease into mice and the way we're doing it is by introducing the human oncogene through a retroviral vector. So the first thing to do in order to introduce this, this human oncogene into mouse cells is to make the virus itself. The next thing to do will be to actually tighten the virus.
So this is my plate of stock, 2 9 3 T cells. I'm gonna have to wash the medium away with PBS 2 9 3 T cells are adherence cells, however, they're not super a adherent, so every movement has to be quite gentle. Once it's washed, we are going to replace the PBS with trypsin.
In order to ize the cells out of the plate, we need to place the plate back in the incubator for about three minutes. So after two three minutes, the cells are actually in suspension. We are going to add a few more ml of regular medium.
This is going to neutralize the tripsin. At this point, we're populating up and down in order to be sure that all the cells are nicely in suspension, we're passing the suspension to a 50 ml tube and to count the cells in presence of triple blue and use a hemo cytometer to count it by hand. So I'm doing my dilution now as calculated and now I'm going to plate three 10 to the six cells per plate.
In four ml medium, we are making sure that the cells are well plated and we are going to put the plates back in the incubator for 16 to 18 hours, which is usually the next morning. So we are now 48 Hours after GFP virus infection. The chloroquine is used in order to increase the uptake of the DNA that we're gonna put on the cells.
We gently sucked the media away from the cells, which have now adhere to the plates gently on the side of the plate. At this point, we are going to replace the plates back in the incubators for about one hour. Meanwhile, we are going to prepare our DNA mixture to apply to the cells tier one that is expressing a neomycin resistant gene and the other one that is actually expressing GFPI am using XI DNA in order to control for the entire transfection.
Finally, we're adding calcium chloride. The use of calcium chloride greatly increases the transfection efficiency. We are now one hour later, We took about the plates with and chloroquine medium and we are going to add our transfection solution.
The last step that we have to do is to actually add one ml of two x sterile HBS while adding the H two XHBS solution. We need to vortex the tube in the same time and go slowly drop by drop. This is to avoid precipitations, immediately distribute one ml of solution to the plate of interest and we are doing that again drop by drop.
At this point, the 2 9 3 T cells are quite sensi sensitized. Because of the chloroquine medium that we're having in. We need to have the all the area covered and to move the plates as little as possible, labeled the plate with the construct that you put on and repeat for the next construct.
At this point, replace the plates in the incubator for seven to 11 hours. At least seven, but no more than 11 because the cells are very sensitized because of the chloroquine and we will need to Change medium after this time. We are now about 30 Hours after transaction.
This is our last change of medium at this point, the 2 9 3 C3 T cells have reached confluence. We have, again, to be very gentle when applying the new medium on the side of the plate. I am using 10 ml syringe with a needle.
I'm going to replace the needle with a filter, so I'm mixing the medium well at this point we have collected our virus and we can place it into minus 80 degrees until used before using it. The first thing to do is to actually tighten the virus. There are several sensitive steps while making virus, and the first one of them is the right concentration of 2 9 3 T cells.
We are plating them at three 10 to the six cells plate because after 18 hours they will be about 80%confluent. That still leaves them the time to grow more, so therefore 24 hours after transfection they will be 100%confluent. It has been shown that this is quite important for an excellent virus titer.
The next step is the use of chloroquine and using chloroquine actually sensitizes the 2 9 3 cells and all the changes of medium have to be done very gently in order for the the entire sheet of 2 9 3 T cells not to lift up from the plate. Finally, the DNA that you're using to make virus has to be extremely clean. We are using C banded, DNA.
Once we have our virus, we keep it in minus 80 degrees. We are usually just following it once by each fo will decrease the tighter of the virus two times. Therefore, we like to freeze the virus that we did and therefore forward before we are doing the actual titer of the virus.
Depending on the viruses, we may have either GFP expression with the virus to track the actual infected cells, or we can have neomycin resistant. And it is very important to actually have the titer of the virus before using it in order to be able to match between experiments or between different mutants.
