Towards the end of the semester we were able to get a new antibody and tested it on the TORC 3A mutant. It took us a while to be able to test the new antibody because the cells were not growing properly and we kept having to delay plating them because they were not ready. Twice we had to bring up fresh cells to see if the new ones would grow better. Since the fridge in the cell culture room had gone out during one of the weekends, this damaged our media and reagents. Once we got the new media in and made new media for the cells, the cells started growing properly again and we were able to carry out one experiment.
We found Tax and TORC3A in the nucleus which agrees with our hypothesis and we are currently preparing another immunofluorescence experiment with the mutant again to confirm this. The last immunofluorescence the result were not very clear because the microscope was not working properly and beam was very weak. On top of that, some of the cells had been washed off the slide. Hopefully tomorrow when I look at the slides the results and images are good and we could therefore move to the next phase of the experiment which is transfecting with TORC and Tax and than seeing the localization of these under the microscope.
Prof. Isaacson and I have been working on immunofluorescence this semester and we have completed three sets of this. For the first experiment, the cells were plated too densely and therefore, when we did the staining and view them under the microscope we could not see individual cells and were only able to see clusters. We repeated the experiment once again and this time plated a smaller amount of cells. The results were better and we were able to determine which dilution of antibody for Tax gave the best signal with the least background. For the TORC antibody our results were not as accurate as the slides were murky and once again not having individual cells on the slides hinder us from making any accurate conclusion on which dilution worked best.
The third experiment consisted of just TORC3A (mutant) and two different type of antibody against it: a mouse and rabbit. The purpose was to compare the signal of the mouse vs the rabbit and the amount of background with each. Since the Tax antibody is a mouse we are not able to use a mouse antibody against TORC because they are both viewed under the same resolution in the microscope and therefore, we will not be able to tell the difference between the two when conducting an experiment. For this experiment, I went with an even smaller amount of cells per wells and we did see an improvement on the slides but they were still not individual cells. We were able to conclude from this experiment that the rabbit antibody was not ideal for the experiment as it gave a weak signal. While trying to take a picture of one of the slides with the rabbit antibody the microscope was not able to pick up the signal of how weak it was. Due to this, Prof. Isaacson will be ordering a new antibody to use against TORC and then repeat the immunofluorescence with the right antibody and dilution against it.
For the fall semester, we have made very little progress in generating the human T cell leukemia virus reporter cell line. We tried different methods in the lab including the modification of our vector. After the modification, we performed transformation and grew cultures to see if the bacteria cells contained our insert. If they were positive our modification was a success. We did this procedure three times and the three times we had no success. Either there were no colonies or the very few satellite colonies that did grow when ran on the gel would give the bands we had seen in the past. As an alternate, we also tried to pop the multiple cloning site out on a 2% agarose gel and than digested one of the mini preps to compare to see if there was a difference between the insert and the multiple cloning site. Once again, we did not see anything. We observed the same linearized bands that were present in the past digestions in the summer and in the beginning of the fall semester. After several attempt and different modification, prof Isaacson and I decided that our vector might be the source of the problem and she is going to try to get a new vector for us to work with in the spring.
As the second portion of my project, I did immunofluorescence to determine the localization of Tax and TORC3 in the 293 Cells. The last immunofluorescence I had performed was back in the summer and I lost the art and the technique to do this experiment. I made several errors when performing the protocol and the results at the end were inaccurate. When viewed under the microscope there was a lot of background on some of the slides but the major problem was that there were barely any cells on the slides. Apparently, most of them were washed away and the antibody that I used to stain I diluted it in water rather than PBS-GC. This along with many other errors along the way contributed to the poor results obtained. Due to this, I will be bringing up new cells to do the experiment on them once again to obtain data that is accurate.
As part of my research, I work with HTLV which is a retrovirus that is related to HIV. HTVL is known to cause an aggressive form of leukemia and lymphoma in patients who get infected with HTLV. During the early stages of infection the virus expresses its gene to establish infection. The purpose of my study is to generate a human T cell leukemia virus reporter cell line that will than be used to examine the viral gene expression of HTLV under different experimental conditions. The overall goal of generating this cell line is to examine the expression of the HTLV gene in the presence of Tax and TORC 3 proteins. The goal of our study is to use this cell line to determine if TORC 3 mutants are constitutively active or inactive and increased or decreased. We are also looking to determine the localization of TORC 3 proteins in the presence of and absence of Tax using immunofluorescence. In order to achieve this, we will be using different method in the lab. During the summer, we used traditional methods of cloning to try to generate the cell line with no success. For this fall, we plan to modify our vector so different restriction enzymes could be use in attempt to get our cloning to work. If our cloning is a success, we will transfect reporter cell line into our T 293 cells and use these for further experiments.