The Human T-cell leukemia virus (HTLV) is a retrovirus that is known to cause cancer and other diseases in adults who have contracted it. The goal of the research this summer was to generate a reporter cell line that would then be used to study viral gene expression under various experimental conditions. Different methods were used to try to generate the reporter cell line. PCR was performed using a plasmid containing HTLV-LTR (promoter) as the template. The PCR product was then digested, ligated to an appropriate luciferase reporter vector, transformed and mini-preps performed on subsequent colonies. A 1.5 kb band appeared consistently on the gels that did not match the size of the insert. After several failed cloning attempts, we developed an alternative cloning procedure-using adaptors to add restrictions sites to our vector. There was only time for one attempt at this new technique that yielded no results. For the upcoming school year, this technique will be performed once again in an attempt to yield colonies that are positive for the insert.
As part of our final project for the summer, immunofluorescence of Tax and TORC proteins was carried out in HEK-293T cells. It is known that Tax activates HTLV through transcription factor and that it physically interacts with all three members of the TORC family. In previous studies, it was shown that TORC could inhibit Tax activity. In order to further explore this, the 293T cells were transfected with HTLV Tax and human TORC proteins, then stained with two different antibodies to the proteins. Two different secondary antibodies then had to be used (one from mouse and the other one from a rabbit) to identify Tax from TORC using the fluorescence microscope. TORC was stained with a 488 nm fluorescent antibody (GFP), Tax with a 594 nm (Texas Red) antibody, and a DAPI stain was used to visualize the nuclei Eight different experimental conditions were viewed under the microscope 1) negative control 2.) Tax 3.) TORC1 4.) TORC2 5.) TORC3 6.) Tax + TORC1 7.) Tax + TORC 2 and 8.) Tax + TORC 3. By using the fluorescence microscope the localization of TORC with regards to Tax was hoped to be observed however the TORC staining was weak and further studies will be performed during the fall semester to obtain better staining and further understand the relationship between the two proteins.
Working in the lab during the summer on a one-to-one basis with Prof. Isaacson was an exciting and valuable experience. I was able to gain knowledge but most importantly gain independence while working on the bench. Doing calculations on a day-to-day basis to split the 293T cells and prepare/modify recipes was a great way to practice my math skills. While working on this research project I had to be persistent and do a great deal of repetition to diagnose what was producing the 1.5 KB band on the gel. Single digestions, using different buffer, and sequencing were used to try to come up with an answer. While trying to come up with a possible explanation, I learned different techniques and by the end of the summer, I knew what everything in the recipe was and how it worked with the other components. Many protocols became second nature to me and I was able to complete them more efficiently.
One of the most valuable lessons I learned from this project was to ask question after question until I was sure I was doing everything right and how to do research on my own. For example, searching for the different enzymes and their percent of activity in different buffers. One of the most memorable experiences I have from working on this project was when I realized I had misread the labels and was using the primers instead of the enzymes for the digestion. This taught me to read the labels carefully instead of relying on my own memory. Another mishap in the lab was when I put glass tubes in the centrifuge without the proper adaptor and two seconds later I heard the shattering of glass and lost my overnight bacterial cultures. These and many more mishaps helped me achieve knowledge that I was not able to gain in a class setting in my past lab sections during the semester. This research project has prepared me to work at a different level for the upcoming fall semester.