Hello and welcome back!
When researchers want to discover or design a drug that treats a certain disease, they also want to know why that drug is effective and how it affects the cell. To make sure that our experimental protocol gives us this information as we try to find treatments for RBM20 induced DCM, we want to incorporate 3 aspects into the protocol:
- Visualize RBM20’s functionality (whether it works or not)
- Visualize where RBM20 is located relative to the nucleus
- Visualize RBM20 expression
This experimental protocol will be performed on cardiomyocytes, or heart cells, that we grow in the lab from patient-derived stem cells. Essentially, doctors and researchers collect skin samples from patients and turn those skin cells into induced Pluripotent Stem Cells (iPSCs). When we receive these cells in the lab, we’re then able to turn them into beating heart cells. Since these cells originated from patients, they are a very close representation of heart cells inside that patient. We’re interested in studying DCM in patients with a mutated RBM20, so we get two “kinds” of cells: those from patients that are healthy and from those who have a clinically significant mutation in their RBM20 gene.
I’ve spent the past two weeks beginning my heart cell differentiation, which is a month-long process that involves adding growth factors and closely monitoring/controlling the cells’ environment. I’m excited to see when they’re finished (probably next week), and I’ll definitely attach a small video of my very own beating cardiomyocytes in a dish.
Furthermore, I also created my RBM20 activity reporter (#1 on our protocol to-do list). First, I obtained a plasmid (a circular piece of DNA) that contains the gene for Luciferase, a protein responsible for a firefly’s characteristic glow. However, I modified this plasmid by inserting a short sequence of DNA that interrupts the luciferase gene. When RBM20 is functional, it is responsible for removing this interrupting sequence of DNA, which results in functional luciferase molecules being produced by the cell. But, when RBM20 is mutated, this process does not occur, and the interrupting sequence of DNA causes the cell to produce a form of luciferase that does not glow. With this tool, we are now able to detect whether the RBM20 is present inside a cell is mutated or not by measuring how much luciferase is being produced. After 3 weeks of work, I finally produced the plasmid I want to create – free of mutations and ready to be used!
The previous paragraph outlines an experiment that theoretically should work; however, before conducting an experiment on precious cardiomyocytes, we tested them out on a type of kidney tumor cells called HEK293T cells. These cells are easy to grow and very resilient (unlike heart cells) and so we are able to use them freely to test my plasmid. To test my creation, this week I transfected HEK cells that I had grown, which essentially means that I poked holes into the cell membrane and tried to have my plasmid enter the cell. Unfortunately, since the transfection process is very taxing on these cells, I was unable to insert my plasmid into the cells without them dying, so I plan to try again this week after altering some of the experimental conditions.