Week 3: CRISPR KO, Mouse irradiation - Alexandra Kot

 2026.06.15:

Today was spent mostly preparing to share my research background in lab meeting tomorrow. However, I also did spend time splitting the cells for the CRISPR knockouts which we will be completing tomorrow. While not knocking out the TREX1 gene, this is a great opportunity to practice the procedure before the TREX1 sgDNAs I ordered show up to lab. 

 2026.06.16:

Tuesday meeting and Demaria lab presentation day! Today the cohort was visited by Dr. Matthew Greenblatt who shared with us a very curated crash course of various bone topics. We covered the developmental stages of skeletal cells, looking into topics like gastrulation and ossification, as well as the development of various structures within the skeletal system like cartilage, the neural crest, intervertebral discs, and the various organic structures and inorganic structures that contribute to bone strength. He also highlighted some recent results from his lab that have challenged the previously accepted dogma associated with mesenchymal stem cells and instead outlines how distinct populations of stem cells with more constrained differentiation pathways govern different regions of the bone. 

I also was asked to present today in Dr. Demaria's lab meeting to share my research journey. I took everyone through my introduction to polymer nanoparticles in Dr. Laura Bracaglia's lab and outlined how that brought me to Battelle during my internships. I also was able to provide a deeper dive into my lab at Cornell under Dr. Harimoto and demonstrated the therapeutic applications of engineered bacteria. Most importantly, I provided an brief outline of my potential thesis project and highlighted how there could be an opportunity for a longer-standing collaboration between labs. I am hoping that, over the course of these remaining 6 weeks, I will be able to outline a few projects that could take advantage of the micro-engineered living material platform and ability for sequential/combinatorial delivery to enhance the abscopal effect through the delivery of certain immunomodulatory molecules. 

Another large part of today was spent observing mouse irradiation. As a part of my week 1 work, I was able to observe the injection of a p53 knockout breast cancer cell line (TSA) into around 40 white mice. Now that the tumors have been able to grow for about two weeks, it was time to begin the various treatment regimens, one of which involved the use of irradiation. Mice were placed in an isoflurane chamber to anesthetize them. Later on, they were oriented on a plate using wooden dowels and tape, as shown below in Figure 1B. The placements of these dowels allowed for only the tumors to be hit with radiation, rather than the whole mouse. As shown in Figure 1C, two mice were able to receive treatment at once in the irradiation machine. 

Figure 1: Photos from mouse irradiation A: Mouse standing cutely. B: Mouse oriented on irradiation plate with isoflurane anesthetic face mask. The tumor, which has grown for two weeks post-injection, is isolated on the plate using wooden dowels. C: Two mice loaded into irradiation machine with isoflurane chamber. 

 2026.06.17:

Today was more of a reading day. I focused my reading on a textbook Dr. Demaria shared with me: Cancer Immunotherapy Principles and Practice by Butterfield, Kaufman, and Marincola. 

But today I did do two CRISPR knockouts of the TLK1 and the MAVS genes in a p53KO TSA cell line. For something that is such a massive breakthrough for gene editing, the process has been made to be so very simple, boiled down to just making two master mixes, which are later combined and then added dropwise to the cells of interest. TLK1 is the Tousled-Like Kinase 1 gene, which encodes an enzyme that is used for DNA repair. MAVS is the Mitochondrial Antiviral Signaling gene, which makes a protein that is critical as a switch for the body's innate immune system. The plated cells are shown below in Figure 2:

Figure 2: TSA p53 KO cells in two wells, one with added sgRNA MAVS and another with sgRNA MLKL. 

Given the remaining steps of the protocol, on Friday the cells should be ready for cloning and will then be ready on Monday or Tuesday to identify wells of single cells. 

 2026.06.18:

Nothing much to report on today. I spent most of today doing a deeper dive into the literature behind TREX1, especially with papers published in Dr. Demaria's Lab. 

 2026.06.19:

Happy Juneteenth! 

Today I came in to do single cell cloning for the CRISPR KO lines I made on Wednesday! Pretty simple process. We cannot guarantee that all cells now have the KO gene. Therefore, cells are split and loaded, ideally, one per well. So that when they grow, we can guarantee that every cell in that well has the same genes. Therefore, when we run a western blot to see whether these cells can produce MAVS or TLK1, we know that the results are representative of the whole population of that clone. 

Though not a hard process at all, I am happy that I am able to practice before my sgRNA comes in. The protocol involves trypsinizing the cells, making serial dilutions to get 1cell/100uL, and the making two 96 well plates. Over the course of the next week I will be able to identify wells where only one cell population is present and run a western blot to confirm that the CRISPR KO was successful!


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