Jerry Wang, Week 2, Starting my own project: molecular cloning of CD3 epsilon

Week 2:

                My P.I. has arrived this week, and she is one of the nicest people I have ever known in my entire life. During the lab meeting on Monday, she even brought us chocolate from London (the best chocolate in the world, according to my P.I.). Together, my P.I. and I had discussed my personal project of the summer: molecular cloning of CD3 epsilon protein. Obviously, our lab’s main goal is to find an effective way to achieve full relapse-free remission in our patients that are suffering from cancer and the instruments we chose to achieve this goal is one of the hottest area currently in immunotherapy: CAR-T cell. The general mechanism behind CAR-T cell is fairly simple. Through electrophoresis or retrovirus, scientists are able to transfer large quantity of mRNA that encodes for CAR receptor into ordinary T cells. Through CAR, we are able to redirect T cell’s specificity against the antigen that will activate the CAR receptor. In other word, through CAR, we are able to “teach” T cell to react much stronger against designated antigens such as CD19 or CD20 that are expressed on the surface of B cells. So, there are many ways to stimulate T cells to grow, including artificial antigen-presenting-cells and magnetic beads. But artificially made antibody that bonds to CD3 epsilon protein on the surface of T cells also has great efficiency in stimulating T cell growth. Therefore, in order to estimate which stimulant will maximize T-cell growth, my lab ordered a large quantity of CD3 epsilon specific antibodies from a biotech company. Now, the biotech company’s job is to inject CD3 epsilon into mice’s blood, which will then cause an immune response from the mice. Then, they will extract the antibodies from the mice. Nevertheless, they will not ensure that these different types of antibodies will work or even bind to our target. Therefore, it is our job to test the effect of these antibodies and choose the one that will stimulate T cell growth and use them when we culture T cells next time.

                In order to test these antibodies which, we will receive in the future, we need to make targets for them, and this is where my project come in. If I successfully clone CD3 epsilon, we will then able to inject their mRNA into K562 human cell line, which will then be expressed on the surface of these cell lines. As negative control, we would have a pure K562 human cell line with no modification. Then we will use flow cytometry to determine whether these antibodies bond to the CD3 epsilon on the surface of the cell line. Then, we will pick out the antibodies which bonded to desired target and test their effect of T cells one by one until we find the right antibody. Anyways, this is a really time-consuming work flow and will probably take more than 6 weeks to complete, but I will try my best to get this project as close to finish as I can.

                So, first thing I need to learn is primer design. My lab member Allie reverse transcribed a large quantity of mRNA extracted from T cells of a canine patient back to DNA. Since the mRNA was extracted from the cell fluid of T cells, there is an extremely high probability that the mRNA encoding CD3 epsilon will be in those samples. Since I could easily find the DNA sequence of CD3 epsilon on Nucleotide, I could design a primer that will specifically binds to that DNA sequence. With the help of such primer, I would be able to run a PCR and get a large quantity of DNA I needed. There are a lot of elements, such as melting temperature, binding site, the length of the primer, GC content at the beginning and the end of the desired sequence and etc. involved in designing a primer and they looked quite intimidating to a lab novice like me. Nevertheless, my lab’s Post Doc, Kazim, was a great mentor in designing such primer, and with his help, now I could confidently say that I am a “master” at primer designing. Kazim sent the sequence to the biotech company and now we have to do is just wait till the primers come in next week.

                I also went to China town to eat Korean BBQ and a nearby Japanese restaurant with Steph, Ping and Trung, it was great fun to see familiar faces around Philadelphia and we had a great time. I wouldn’t go into the details of these thing though since they are not really research-related. But it was great to know that everybody enjoyed their summer while at Penn and they were all making progress with their individual project.