Katie Munechika / Dr. Ethel Cesarman - Week 2

This week, we received H1E and H1C knockout cell lines from a collaborator, which were created a few months prior using CRISPR-Cas9. Unfortunately, the majority of the cells were dead, so I have been working on trying to save and expand some of the cells that were still viable. Once these are at a proper viable percentage, I can extract the genomic DNA and sequence them with Sanger sequencing to confirm that they are truly H1E and H1C KO.

Regarding the validation of the V5-tagged H1E cells, we received a new V5 antibody, so I stripped the western blot membrane and redid the antibody incubation and imaging. Although the β-actin antibody I used last week worked, there was some obvious non-specific binding, so I also tried a new actin antibody to try to reduce that. Sadly, this didn’t work at all and this time I could not see any bands. It is possible that the membrane was not stripped well enough to allow the new antibodies to bind. Next week, I plan to repeat the entire western blot process, starting from lysing the cells. Hopefully, with a new membrane alongside the new antibodies, it will finally work.

I have also been helping with another project that is currently testing effects of two drug treatments on different histone markers of primary B cells. In order to do this, I have learned how to perform chromatin immunoprecipitation (ChIP), which consists of fixing and staining the cells, sorting them by flow cytometry, performing a nuclear extraction, and then purifying the DNA. Once the DNA has been extracted and purified, we can perform quantitative PCR.

On the clinical side of things, I had the opportunity to shadow Dr. Morales, an infectious disease specialist and was able to sit in on some appointments. One patient had an infection that caused inflammation and swelling of the toe that began after a tendon-related foot procedure they had undergone. The inflammation was to the level that it was difficult for them to walk and they were in constant pain. The patient had tried various different antibiotics per the recommendation of their podiatrist in the months leading up to this visit, but the infection persisted, which is the reason they were referred to an infectious disease specialist. Dr. Morales ordered an MRI to better understand how deep the infection was, whether it was just in the soft tissue or if it was potentially in the bone. Dr. Morales also wanted to obtain a deep culture of the infected area from a podiatrist, based on the imaging results, in order to prescribe a more specific antibiotic. Since there was no discharge from the infected area, it was not possible to obtain a proper culture sample at the time of the appointment, meaning a deep culture was required. I found it particularly interesting to observe Dr. Morales’ thought process and how they worked with the patient to try to figure out what may have initially caused the infection, as well as decide the best next steps for addressing the issue. As an engineer, bedside manner is not something that I usually think about, but it was very eye-opening to see how Dr. Morales was sympathetic to the patient (who was clearly distressed since this condition greatly limited their mobility) and made them feel understood, while also collecting the necessary information for diagnostic purposes.