Alexandra Kot, Week 2: sgRNAs and Clinic

 Monday: 2026.06.08

Today was a big day for the start of my summer immersion project. After spending last Friday looking for TREX1 knockout sgDNA sequences, Dr. Shahbandi and I chose three of which to order from ThermoFisher. Part of ordering was learning to confirm sequences using BLAT (BLAST-like Alignment Tool) to check for the presence of the sequences in the mm39 genome (mouse genome) and look for any off target effects which could result from the sgDNAs. This was especially essential for this set of sgDNAs, as the lab had tried to make a TREX1 knockout before, but was previously unsuccessful. The possible solution is to increase the number of sgDNAs used, but the caveat is that there is a much higher chance off an off-target knockout. As this is all new to me, I have been trying to do a deeper dive into CRISPR so I can better understand the experiments I will be performing. 

I also was able to start a western blot on cells which were multiple days post irradiation. This included a lysis step to harvest the inteferons.

We ended last week with the imaging lecture with Dr. Prince. Though my background does not have anything to do with imaging or with orthopedics, Dr. Prince outlined the imaging concepts well and worked with us earnestly to build our confidence when it came to making conclusions from CT, MRI, and X-Ray imaging. He shared with us that we should focus on larger concepts before narrowing in to the imaging. Starting first with the body part, then the image type (denoting what kind of tissue/structures were being highlighted), points of recognition, and then finally narrowing into any abnormalities. With help from Dr. Prince, I was able to identify a deep vein thrombosis. 

Tuesday: 2026.06.09

During this weeks meeting, Dr. Elemento, the director of the Englander Institute of Precision Medicine, presented on the development of precision medicine through using multiomics, conclusions based off of tissue architecture, and the development of patient organoid avatar to assess therapeutic outcomes. We were also able to meet Dr. Min. It was great to hear his background and his views on healthcare economics, preventative medicine, and personalized risk assessments. It was refreshing for him to admit that there are problems with American healthcare and how he has plans to change them. 

After the weekly meeting, I was able to catch the end of Dr. Garate's data presentation at Dr. Demaria's lab meeting. 

I was able to help with a prior experiment which is performing a similar CRISPR knockout in the TSA cell line. At this point of the experiment, the knockout has already occured, the cells had been separated into 96 well plates for cloning. I aided with clone identification and with trypsinizing the clones for passaging into 12 well plates. 

Wednesday: 2026.06.10

Today was mostly a reading day. I was focusing on two papers from the Demaria lab: TREX1 regulation of RT tumor immunogenicity and a patient clinical trial which combined RT with ipilumumab. I was also able to help the lab technician, Maxine, set up and run an ELISA with samples we had collected last week. 

Thursday: 2026.06.11

Today was very exciting! It was my first clinic day with Dr. Demaria in Pathology and Laboratory Medicine. With one of the other medical students, we looked at many different H&E slides of breast tissue from a variety of different patients in a very interesting sign-out room. On a meeting table, the microscope was controlled by one user, with multiple arms extending out to different chairs so that onlookers could adjust the eye piece as needed. We reviewed cases for a few hours, with nothing terribly of note but some samples seemed incomplete so more levels were ordered. The final case was very interesting. A patient opted to receive a double mastectomy after identification of a small carcinoma (<0.5cm) in one breast. This led to a deep discussion as to the health, mental, and social motivations for why a patient would elect for a double mastectomy which might not have been recommended by a doctor. 

I was also able to get into a gross room. Gross meaning macroscopic, but definitely not the most enjoyable room to be in. Here, they organized surgical excisions and cut them into small cassettes for further staining and microscopic analysis. This is where I saw the following:

Figure 1: Two breasts, removed via mastectomy, which had been sliced to ensure adequate formalin penetration. 

I was also able to aid in the second half of a western blot: the transfer step. As I will be doing many western blots during my immersion project, it was great to see the process so I could know what to expect.

Friday: 2026.06.12 

Today was more clinic. Dr. Demaria had remarked previously that Fridays are normally her most busy clinic days, but today there were only just as many slides to review as yesterday. However, these cases were more clinically interesting. While many cases did present as benign (papillomas, fibroadenoma, apocrine metaplasia), there will still many cases which were malignant. These cases mostly consisted of ductal carcinoma in situ and invasive ductal carcinoma. I was hence introduced to the grading system used for invasive ductal carcinomas. 

The remaining of the day was spent preparing for lab meeting next Tuesday where I will be presenting on my research background and thesis plans.


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