Research has always been of interest to me, and it was a major part of my decision to study biochemistry, cellular and molecular biology here at UNH. Naturally I was excited to be accepted into the Research Experience and Apprenticeship Program (REAP) this summer and begin my project on red blood cell differentiation and a new protein that may play a role in myeloid cancers and other diseases.
My project was to validate the effect of knocking-down a gene (lowering its protein expression to a very small amount) for a novel protein, C1ORF150, in hemopoietic stem cells. Previous experiments have suggested that this protein governs the proliferation and differentiation of red blood cells, which has a wide range of correlated health issues such as myeloid leukemia, melanoma, juvenile arthritis, and even inflammatory bowel disease. Without C1ORF150, the cells divide unchecked, and they do not differentiate into the mature cells they are supposed to become. This is a hallmark of cancerous development, which leads us to believe that this protein plays an important role in the signaling of the normal cell growth cycle.
At the start of the program, I knocked-down the gene that codes for our protein and started the first of many growth assays to determine the optimal dose of erythropoietin to keep the cells alive. Erythropoietin is a crucial growth factor hormone that is necessary for red blood cell production. In normal cells it appears that when there is no erythropoietin to signal for the production of more red blood cells, C1ORF150 stops the cell from proliferating.
We studied a UT7epo line, which is our most used cell line. A cell line is like a lineage, where all the cells come from a select few from a patient. The first cells of our cell line were isolated from a patient in Japan who was experiencing a rare type of leukemia where the cancerous cells almost fully differentiate into red blood cells before they stop their development. This is unique because most cancerous cells don’t reach these later differentiation stages. The UT7epo line is dependent on erythropoietin for survival, which sets it apart from some other types of cancer cells that can develop without erythropoietin.
On a weekly basis, experiments normally spanned three or so days. To account for this, they were set up on a Monday or Friday so that we weren’t coming in to check on them at midnight on a Thursday or weekend. To conduct a growth assay, we performed cell counts with a hemocytometer, centrifuged and adjusted cell densities by volume, and needed to consistently pipet equal volumes of cells with varying levels of the erythropoietin hormone.
It was important for our growth assays to be optimized such that we could see the full range of effects from so little erythropoietin present that the cells are dying, to more than enough being present. This was crucial for when the lab performed the knockdown in the very expensive hematopoietic stem cells in late August/early September, as the cells would die if there was too little erythropoietin. Following the collection of data, which normally included hemocytometer counting, imaging, or some other analysis method, we summarized and shared our results.
My activities changed weekly as experiments progressed, which kept me on my toes. Fortunately, there was also some consistency to my schedule. Each day I met with my mentor, Don Wojchowski, and a post-doctorate researcher to go over the current experiments, past results, and check in on what was going on in everyone’s lives. I was constantly asking questions and looked forward to the passionate answers and tangent discussions that often occurred. Additionally, I always checked my cell cultures and replated them at a lower density if needed so they were healthy and growing and could be used for our experiments.
Beyond the physical lab work, I spent a lot of time researching the techniques, protocols, and knowledge behind the experiments we were doing. I followed a lot of rabbit-holes such as the history of blotting techniques and fun facts about their origins—like that the “Southern blot,” a form of fragmentation analysis, is named after Mr. Southern. What is humorous is that the blot naming system is framed by the cardinal directions, i.e. the Northern blot and Western blot, despite none of the subsequent developers having directional last names.
I wanted to learn as much as I could, so I also tagged onto other projects in the lab. This allowed me to learn more than I could ever wish for, and it added an extra layer of complexity to my schedule. I wouldn’t have been able to do this much without the support of the lab. They have been so encouraging and passionate about my experience and education, and I can’t thank them enough. I never imagined how many meaningful connections I would make and the life lessons I would learn beyond the science. I am planning to continue my project past the end of my REAP experience and am looking forward to doing research during the school semester.
