Katie Munechika / Dr. Ethel Cesarman - Week 3

 In tackling the problem of the unsuccessful western blot, I decided to re-verify the DNA sequence to ensure that the V5 tag was inserted correctly at the N-terminus of the H1E isoform. To do this I extracted genomic DNA from the cells, performed PCR, and sent the product for Sanger sequencing. By aligning the sequences and checking the chromatogram, it seems that the V5 tag is present in some of the alleles, but not others. I plan to try the western blot again with more cells, as well as use some different antibodies.

In the meantime, I have been designing a CRISPR Cas-9 experiment to generate other cell lines with V5-tagged H1 isoforms. This has been a bit of a learning curve for me, since I have no previous experience with CRISPR. I have looked into a few papers that have optimized CRISPR protocols for their own purposes. I’ve also been testing out some of the online tools for designing guide RNA and donor DNA templates to gain a clearer idea of how to proceed with this project.

I had the opportunity to shadow Dr. Morales in the infectious diseases department again. One interesting part that I was able to experience was the process for deciding how to treat a patient with latent tuberculosis. It was very intriguing to see how Dr. Morales chose which medication would be best suited for the patient’s specific situation. There are two main medications that are generally used to treat latent tuberculosis, but it was important to check for interactions between the tuberculosis treatments and any current medications the patient was on. For example, the patient was also taking risankizumab, an IL-23 inhibitor, to treat their psoriasis. As an interesting side note, Dr. Morales explained that psoriasis used to commonly be treated with tumor necrosis factor (TNF) antagonists that would target a cytokine responsible for regulating inflammatory processes, however these TNF antagonists can be associated with increased risk of serious infections, such as tuberculosis, because they are not very specific. The IL-23 inhibitor this patient was taking is a more targeted method of psoriasis therapy believed to be a safer option than the TNF treatment. Dr. Morales also had to take into consideration the side effects of each of the medications, as well as which would be preferable given the patient’s work and lifestyle. For example, one of the treatments had potential to affect liver function, so she had to verify that the patient had good liver condition in order to consider that as an option. Even though this was a quick visit, there were many key factors that needed to be considered in order to determine the best way to move forward with treating the patient. These types of experiences really make me think more carefully about the aspects that engineers must acknowledge when developing medical technologies and how important it is to gain a multi-perspective view of the problem, so that key components are not missed.

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