Final Paper

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 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.

 

Reflection

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.

Blog 2

So far this summer we have had no success cloning the reporter cell line.  I performed several PCR’s that would initially generate a positive result.  Yet, after running them on a gel they would prove to be false, as the bands did not match the size of our insert.   After several failed attempts, Prof Isaacson and I prepared and  sent-out a sample to be sequence to determine what exactly were the bands.  The results came back and to our disappointment the bands were “junk”, in other words, they did not represent anything.  Prof. Isaacson suggested cloning using adaptors to add a restriction site.  After extensive research on how to do this protocol, we ordered the necessary enzymes and performed the procedure but did not have any success, as no bands appeared on the gel.  The DNA concentration and ratio were very low (we tested the concentration of each sample) one possible explanation as to why we did not see any bands.

This internship has challenged me in many ways, as it has forced me to see research for what it is: a lot of repetition and failure.   It has also taught me to be patient, persistent, and most importantly that there are more than one way to do certain procedures.   For example, I learned to do PCR one way from my cellular molecular class but while working with Prof. Isaacson, I learned two other techniques.  I have also learned many other skills that have allowed me to work more independently at the bench and to think of possible ways to troubleshoot when things go wrong.  I learned how to do transfection, grow cell cultures with 293T cells, and immunofluorescence.   As an undergraduate student, I was able to perform techniques that other students only read about in books.  Overall, this has been a positive experience that has motivated and allowed me to grow as a student.

Examining The Function Of The TORC 3 Protein In HTLV In Gene Expression Blog 1

This summer Prof. Isaacson and I are working on generating a human T cell leukemia virus (HTLV) reporter cell line that will allow us to conduct further studies throughout the year.   The purpose of generating a reporter cell line is to examine viral gene expression under various conditions using different proteins that affect its expression.   So far, I have performed PCR, ligation and transformation in order to clone the HTLV promoter into a luciferase vector.  Our research seems to be headed in the right direction, as many colonies grew on the experimental plate but none on the negative controlthe colonies that grew on the plates were white in color.  The white color is an indication that our transformation may have been successful, as our insert (PCR product) could have been taken up the bacteria cells.

Working in the lab  one-on-one with Prof. Isaacson has been an exciting opportunity as I am able to ask questions and get thorough explanations.  Observing how Prof. Isaacson carries out certain techniques in the lab has allowed me to “brush up” and become more efficient in carrying out protocols. Even though I have participated in laboratory sections as part of my major curriculum in the past, it cannot compare to the experience I have obtained in this past month.  The input from the other students working in the lab has been an amazing experience too.  I am able to learn from them and they have been quick in giving me tips and assistance when I need it.  Working in a cramped research room (while the biology department is under renovation this summer) and observing how Prof. Isaacson thinks outside the box to carry out certain protocols has expanded my creativity.   For example, we were not able to obtain ice from the cafeteria and we needed ice to carry out our ligation.  Prof. Isaacson at first used Ice Packs and than later decided to buy ice cube trays to create our own stock of ice.   We also do not have a gas line in the research room so we are forced to use an alcohol glass lamp burner, something I had not seen or used before.  Seeing how Prof. Isaacson thinks makes me look around the room twice when we I come across a problem before asking her for help.   Hopefully our research keeps heading in the right direction and we are able to accomplish everything we had planned this summer.