Finishing one project, starting another

The past few weeks have been very busy as we are completing the nucleophagy project and I am transitioning to working on the the ALS one. In order to do this, I have been collecting a large amount of data confirming that the process we are describing, nucleophagy, is the response of the cells to stress and is not an artifact.  In order to do this, I have “knocked down” or decreased the expression of a protein (LC3) involved in the formation of autophagic machinery, which we use as a marker of overall autophagy in the cell. Stressing these cells with chemical compounds that we know increase or decrease macro autophagy or nuclear autophagy should result in a predictable change in LC3 expression. We knock down LC3 expression via transfection of a short hairpin RNA (shRNA) which binds endogenous mRNA in the cell and degrades it before translation can occur. In the experiments I am conducting, we expect that wildtype (WT) cells will show the predictable level of LC3 change in response to treatment, while cells that have been knocked down will have significantly reduced expression regardless (as LC3 is not being translated no matter the conditions). I have had some issues with these experiments the past few weeks, but I am making a big push over the next few days to get at least four separate experiments finished and imaged by early next week. After that, I plan on doing similar experiments but instead using confocal microscopy to create a more qualitative representation of the data produced in earlier experiments.

Macroautophagy and the role of LC3


Finishing this project leads nicely into the work I am also doing with the C9orf72 mutation and its involvement in the onset of ALS and other neurodegenerative phenotypes. Amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease affects roughly 20,000 people per year, and know about the disease because of the “Ice Bucket Challenge” social media campaign. ALS is a neurodegenerative disease that results in death of motor neurons in the motor cortex of the brain. There is currently no way to slow or stop this process, and it is difficult to pinpoint the exact causes. We know the C9orf72 repeat is involved in the development of this disease, but there is little research on the mechanisms of degeneration in a tissue culture system. In order to study this, we need to have some method of introducing the repeat into our cells (either neuronal or cancer cultures). I am currently working to design a virus that contains the C9 repeat and the machinery necessary to infect the cells and cause the expression of the gene. In order to do this, we have to remove the C9 portion of a plasmid we already have and insert it into a lentiviral backbone, which we also have in the lab. The C9orf72 has a very high guanine and cytosine (GC) content, which makes traditional methods of cloning difficult. Instead, we plan on using a technique called Gibson assembly to create the individual parts of the viral plasmid that we can then put together relatively easily. We have recently been running restriction digests to confirm cut sites on the plasmids, and we will then need to make primers for the assembly.

Gibson assembly