Week 5: Clone Expansion, Mouse Irradiation - Alexandra Kot

 Alexandra Kot | Dr. Sandra Demaria

2026.06.29:

This morning we attended a lecture from Dr. Denise Howard, Chief of Obstetrics and Gynecology, titled Addressing Disparities in Women's Health with Tech and Innovation. I was shocked to learn how, despite being a leader in healthcare spending, the US experiences 2.4x more maternal fatalities than Canada and 8x more than Japan. Dr. Howard showed some of the demographics behind fatalities and revealed how Black women are particularly affected. Dr. Howard shared some of her own ideas for solutions, which were centered around technological changes. 

I spent today culturing the single-cell wells into larger 12-well plates. I did not know that transferring a small amount of cells to a larger culture flask could "shock" the cells. So I will have to expand my cells into 12-well plates (skipping 24-well plates due to confluency) and then into 6-well plates. And here begins the dreaded cell culture waiting period. Despite having many (74!) clones, I have to wait to know if the MLKL or MAVS knockouts worked until the cells have been expanded to larger culture dishes so I can run a Western to screen for the (hopefully) knocked-out proteins. 

2026.06.30:

This week's Tuesday meeting involved a visit from Dr. Mert Sabuncu, where he presented on how AI and imaging can help with personalized risk assessment. Dr. Sabuncu outlined a number of projects that are ongoing in his lab. First, for the tracking of disease progression for autosomal dominant polycystic kidney disease, which demands <5% error for making diagnostic decisions. With Dr. Sabuncu's AI system, he achieved a 1.3% error rate with only two minutes of processing time. 

The plan for my knockouts, given how much they have grown over the past 24 hours, is to let them continue growing until Thursday, at which point I can expand them again to 6-well plates. Then, after the weekend, they should be at the point where I can freeze one 6-well for future use and begin the lysis steps for the western blot. 

Most of the rest of my day was taken up by reading. However, I was able to observe another mouse irradiation experiment. These mice are receiving 8Gy radiation over three days. This 8Gyx3 dosing was shown by the Demaria lab to stimulate interferon production due to the cGAS/STING pathways, which is essential for the activation of the immune system. At this level of radiation (as opposed to just a one-time delivery of 24Gy), TREX1 is not expressed. This is vital to the effective stimulation of the immune system, as TREX1 cleans up DNA in the cytoplasm (which is common after radiation), which decreases the level of DNA in the cytoplasm, which is essential for cGAS/STING activation. 

2026.07.01:

Today was mostly a reading day and observing mouse irradiation. I have been getting through more of Cancer Immunotherapy Principles and Practice by Drs. Butterfield, Kaufman, and Marincola as well as through more Demaria research articles. 

2026.07.02:

This morning I expanded my cells as was previously planned. Which took a whopping 2.5 hours. Each of the cell clones in the 12-well plates had to be split between one western blot 6-well plate and one continuing culture 6-well plate, producing a total plate count of 26, half of which is shown in Figure 1. Once these cells grow over the weekend, they will be lysed or frozen in preparation to do a western blot to confirm whether we obtained a knockout. 

Figure 1: Expanded TSA sgTp53 MLKL and MAVS knockout cells in 6-well plates, labeled for Western blot use. 

The week finished with one more final day of mouse irradiation. 


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