Blog #4 Luciferase Assay with TORC mutants

From my last post I mentioned that my CHO reagents no longer worked. For the experiment that I recently just finished, I substituted the CHO reagents with LT1 reagents. As mentioned in my previous posts I maintained my CHO cell line for at least a week before plating them into wells. After plating them into the wells and waiting 24 hours, I transfected my reagents and my plasmids into the cells in the plates. Finally, I performed a luciferase assay after aspirating and lysing the cells to determine their readings.

I tested eight different categories of plasmids which were the LTR, Tax, TORC3, TORC3A, TORC3D, Tax+TORC3A, Tax+TORC3A, and Tax+TORC3D. With these eight categories I receiving my results for 24 tubes. Unfortunately my data did not come out as expected. My TORC3 had an extremely low reading when in previous experiments we had TORC3 having the highest. TORC3D is also supposed to have a lower gene expression than TORC3 but in my results it was shown to be higher. Tax+TORC3 was also unusually low. Tax+TORC3D has higher gene expression than Tax+TORC3. This may indicate a problem during the transfection. TORC3A and Tax+TORC3A seemed to be the only mutant playing its role. It has an extremely high gene expression compared to the others. This mutant is supposed to have a higher gene expression since it is dephosphorylated into the nucleus and is active.

So far my results have not been as expected. This project just shows me how hard research is and how every little thing counts. Especially since my experiments are over a course of three days. Any problems within those three days can greatly alter my results. As for this project, I hope to continue working with Isaacson so I could obtain the ideal results.

Luciferase Assay of TORC3A and TORC3D

At this point in our research project, I was told to do another luciferase assay because we suspected that our stocks were mislabeled. From our last post, we sent out our mutants for sequencing to determine if they were correct. The results showed that we had the right mutants. So we proceeded to repeat the experiment to see what went wrong. I finished another luciferase assay on 2/25.

In order to begin my luciferase assay, I repeated the same steps mentioned from my last post. I had to maintain my cell line for at least a week prior to the experiment. In a week’s time I counted my cells and managed to split my cells into a flask in order to plate them into wells. The next day, I transfected the mutants with the appropriate amounts into each well and let them grow for one more day. The day after, I was able to do my luciferase assay. However, there was a problem. Over the weekend, the fridge shut off and my reagents and media must have went bad. My results from the luciferase assay was not strong enough for me to keep the data.

I was pretty disappointed that I worked really hard just for something that was ruined by something as small but so major as my reagents not being cold for 3 days. However, I learned that with research every little thing counts. Everything has to be as precise and as exact as mentioned on the protocol for the best results or to just succeed with the experiment. Although I was disappointed, I brushed it off and told myself that I’ll just repeat the experiment and get better results next time.

Blog #2: Luciferase Assay of TORCD Mutants

As mentioned in my last post, we wanted to test TORC mutants in a CHO-cell line. Unfortunately, I was unable to start right away because of the move in my department and having to wait for the culture hood to be certified and approved. However, I was able to start maintaining my cell line in early October up until mid November. After maintaining my cell line, I was able to construct a lucfierase assay in late October. Prior to the luciferase assay, I had to split and transfect the cells along with their TORC proteins and mutants. This experiment is a three day process.

After splitting and transfecting my cells, I was able to generate a luciferase assay. The data we achieved looked normal in mutants TORC1D and TORC2D. However, our TORC3D mutant generated a large amount of gene expression. As a reminder, TORCD mutants are supposed to mimic a phosphorylated state which is supposed to have an inactive state whereas our previous TORCA mutants have the opposite effect. In previous experiments, we also have found that TORC3 always show the highest amount of gene expression compared to TORC1 and TORC2. However, as mentioned before, TORC3D has an extremely high and abnormal amount of gene expression for something that is trying to mimic phosphorylation and an inactive state. Therefore, our data is a little off. However, we believe that our TORC3D stock was mislabeled which means it is not really our TORC3D mutant. Therefore, we will send them out for sequencing and check their sequences to ensure whether or not it is our TORC3D mutant and whether we should attempt to repeat the experiment on the TORCD mutants.

Some questions that came up after receiving my data is:
Why does TOR3D and TORC3D + Tax show abnormal gene expression?
If after sending them out for sequencing and confirming it is the TORC3D mutant, is it worth repeating the experiment again?

 

Blog #1: Cellular TORC Mutants Enhance HTLV gene expression

My purpose of my project is to create TORC mutants. TORC is a protein that has been recently found to help activate human T-cell leukemia virus (HTLV). HTLV is known to cause adult T-cell leukemia and lymphoma (ATLL). In normal conditions, TORC activity is regulated via phosphorylation. We are testing to to see whether the phosphorylation state of TORC impacts HTLV transcription. In under normal conditions, the TORC protein is active by dephosphorylating and entering the nucleus. On the other hand, the TORC protein is inactive by phosphorylating into the cytoplasm.

We have successful created two TORC mutants in the summer and want to prove their influence on gene expression. We have a TORCA (serine to alanine) and a TORCD (serine to aspartic acid) mutant. These two mutants were created to mimic the two different phosphorylation states mentioned above. TORCA should mimic a dephosphorylated state and be constituitvely active. This should result in an increase in HTLV gene expression. TORCD should mimic a phosphorylated state and be constitutively inactive. This should result in a decrease in HTLV gene expression.

In order to show how our mutants influence HTLV gene expression, we will first transfect them into a chinese hamster ovary cell line (CHO). If the results show successful data, we will transfect them into Jurkat cell line. Jurkat cells are human T lymphocyte cells that are used to study acute T cell leukemia. These are harder to transfect but we will have multiple attempts to test our mutants on this cell line.