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?

 

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