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.

TORC 3

This semester Prof. Isaacson and I are working with HEK 293 cells instead of 293t cells.  In the past, we worked with 293 t cell line and study the localization of the TORC family and tax protein in these.  HEK 293 is a cell line derived from human embryonic kidney cells in culture and they differ from the 293t cells because they do not contain the SV40 large T antigen that is capable of inducing malignant transformation.  The purpose of working with the HEK 293 this semester is to study localization of the TORC family in the cells and afterwards compare it to the localization of these proteins in the 293 t cells.  We are interested in seeing if malignant transformation of the cells affects the localization of the proteins (TORC).

We are currently doing immunofluorescence to study localization but so far this semester they have all been a fail (3 so far).  The HEK 293 cells do not grow as fast as the 293 t cells and do not adhere as well either.  This has proven to be a problem as they are harder to maintain after every split and they do not adhere to the glass cover slips very well. In fact, I had to dispose of two plates because they did not adhere at all to the glass cover slips. If they do not adhere, I am not able to perform any experiment on them and this has been my biggest challenge this semester.  I am hoping that by treating the glass cover slips with a special chemical the cells will be able to adhere and I will be able to perform my experiments.

Tax and TORC 3/HTLV Gene Expression

As part of my research I will be working with 293T cells and analyzing TORC and Tax localization in the cells.   I will also be analyzing the localization of Tax and TORC in 293 cells and than comparing both of the localization in normal vs. cancerous cells.   The main goal of the research is to analyze the gene expression of HTLV in the presence of tax and cellular transcription factors, which are thought to enhance HTLV gene expression.  HTVL is the first retrovirus that was discovered and it is known to develop an often rapidly fatal leukemia, debilitative myelopathy, uveitis, dermatitis, and inflammatory disorder among others.  The goal of this academic year is to finalize our findings and determine if TORC 3 enhances HTLV gene expression.

 

To study localization we will be using immunofluorescence technique.  For this technique we use antibodies that are specific to tax and TORC 3.  Each antibody will emit a specific wavelength of light once excited allowing us to view it under the microscope. The light is specific to the antibody (red or green) and is only emitted when the antibody is present in the cells, indicating the presence of the protein of interest in the cells.   Prior to staining, we will transfect cells with Tax and TORC, stain with the antibody, and view the slides under a special microscope.  To transfect cells we must grow them in a special media and incubate them in a regulated environment to ensure their growth occurs at optimal conditions.   Many times many trials of immunofluorescence are needed to obtain pictures that are of good quality. I look forward to all the exciting new challenges this academic year will bring.

Blog 4

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.

Immunofluorescence Experiments

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. 

Blog 2: HTLV

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.

HTLV, a retrovirus that is related to HIV

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.

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.