Final Report


Overview

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 spring semester, 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 were 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. Furthermore, we did immunofluorescence to test the mutants created by one of my colleague and confirm that the mutants were working properly. She generated several TORC mutants and I was able to confirm her hypothesis about their localization. I was also able to test out a rabbit polyclonal antibody that was not very successful with the cells. This antibody produced a great amount of background and therefore, different options have to be consider to carry out a co-localization for TORC and Tax.

Reflection

Working in the lab during year 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 and confidence in my own skills.  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. I was responsible for maintaining my own cell line and I learned how to bring them up, freeze them down, and became comfortable enough to make decisions on my own about them. I also learned how to count cells and how to plate them as well as, how to trouble shoot some of the problems we ran into with the cells. Many protocols became second nature to me and I was able to complete them more efficiently.

This project put me in the position to present at the Eastern Colleges Conference, poster presentations, and for my own research with the school. This forced me to speak to others about my project and practice my communication skills. I learned how to present and how to communicate effectively when people would ask me questions. I would constantly get bombard with really challenging questions but because I became so comfortable with my own project, I was able to answer these. This project prepared me beyond the laboratory and gave me a taste of the real world. Moreover, I became proficient at reading and understanding scholarly articles in the science field. Prior to working in the laboratory, I did not feel comfortable reading scholarly articles as I found them really hard to understand but as part of our weekly lab meetings we were required to read scholarly articles and than present them to the team. This was extremely useful as I was learned how to better interpret the data and process what I was reading. Furthermore, I enjoyed being part of a team of students who would look out for each other and became friends that were quick to help when I had a question.

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