Blog Entry 1:
My name is Maya Golob, and I am a rising senior at Harvard-Westlake School in Los Angeles, California. I am one of eight research interns in the seven week CIRM SPARK summer program at Cedars-Sinai. I am working in the Regenerative Medicine Institute under my mentor, Gianni Carraro, in Dr. Barry Stripp’s lab.
I’ve nearly completed my first week in the CIRM SPARK research internship at Cedars-Sinai. With my assigned mentor out of town, I have had many lectures and from the Research Associates about what they are working on and other things that go on in the lab. In four days, I have already learned so much! I want to explain the three main topics I’ve learned: flow cytometry, gel electrophoresis, and CF cells.
Flow cytometry: Each cell has different surface markers. We can select fluorochrome antibodies to bind to a specific surface marker that we want to study, which is essentially labeling the cells because they create a fluorescence that we can track. We then put the cells into the machine and lasers excite them, making the electrons jump to a higher energy state. When the electron falls, it releases energy called a photon (light energy). This allows us to see the colors the cells emit and the machine sends data to the computer for each cell. We can then track which cells glowed certain colors (meaning they are positive for a certain gene), and check to see if the experiment is working all in a matter of seconds.
Gel electrophoresis: First, we isolate the DNA from bacteria following the steps in a kit. Then we add restriction enzyme digestion. With an Nkx2-1 plasmid, we use HindIII as the enzyme to produce cuts with sizes 7001, 1001, and 1011 base pairs. We run the DNA through the PCR machine to make copies of the DNA. Then we stain the DNA and insert it into the 1% Agrose gel we made. The results of the gel electrophoresis tell us that the plasmid was successfully inserted into the DNA if the bands line up with the 7000 band and the 1000 band.
CF cells: These cells are a certain type of cystic fibrosis stem cells. SOX2 is an essential transcription factor for maintaining self-renewal (pluripotency) for stem cells. We can insert this into the cells by creating a plasmid with the SOX2 gene via bacteria. We can then extract the plasmid from the bacteria through a series of steps. Once we have the plasmids, we can insert them into the cells and check it with gel electrophoresis. The cells will first produce GFP, meaning that they glow green. Then the guide RNA, which we made to find the gene of interest, tells CRISPR where to cut. Bacteria use CRISPR as a defense mechanism against viruses because it cuts the DNA of the virus. Scientists found a way to use it to edit the DNA of all cells, including human cells. Then, natural transcription will insert the plasmid loop into the DNA, which takes about three days. If the plasmid is digested successfully, it will leave the SOX2 gene and blue fluorescent protein (BFP). Through the fluorescent microscope and flow cytometry, we can see if the cells glow blue. If they do, we know that they contain the plasmid we wanted.
In these four days, I was also able to practice sterile technique while culturing mesenchymal cells in the tissue culture room that has the fume hoods. I was able to practice my pipetting while making more media, a colored fluid that the cells live in. I look forward to working with my mentor when he returns and I’m eager to start working on my project for the summer!