Lauren Donato Week 4 Mason Lab: CD 21 Cloning Sucess

Week four was a 4 day week, due to fourth of July! I hope everyone saw some fireworks and had  a nice day off. Now that the vaccine was frozen down from the B Cell lymphoma project I was working on, I began a cloning project for the lymphoma study. Adoptive immunotherapy (AI) using autologous, re-directed T cells to recognize and kill tumor cells is a powerful strategy to target primary, metastatic and relapsing disease. In this approach, T cells are genetically modified to express tumor-specific chimeric antigen receptors (CAR) that endow these cells with MHC independent antigen recognition capabilities and antigen-dependent activation. For my project I am using a retroviral approach to transduce canine T

cells with CD21 targeting CAR constructs that contain the canine 4-1BB and CD3ζ intracellular signaling 

domains. CD 21 is a very long gene on the CAR construct, but is not the only gene. Our lab has successfully coded and transduced cells for other genes on this cell line, including CD19 and CD20, but CD21 is not completely coded. The first step of my project that I completed during week four was to properly amplify the CD21 gene. The lab ordered a special enzyme for me called Longamp, which is supposed to amplify long genes. Instead of giving me a procedure I was given the basic materials and had to optimize the best way to clone this gene. I used various amounts of cDNA template for one of my PCRs, and then kept that amount of cDNA standard for the next round of PCR. I also tried setting the PCR machine to a gradient during one step of the cycle to see what worked best. By keeping constants and being creative I optimized a successful PCR. 

These were my gel results from varying the amount of cDNA template from least amount to most so I picked the brightest band to do a gel extract and retrieve the DNA to sequence.

completed a gel extract of band 4 to get the DNA, this was fun I literally cut the gel

My PI was away while this was happening and prior to her leaving I got this message: 

I did not burn the lab down

By gel electrophoresis and DNA sequencing I figured out that I was able to amplify the gene. I presented the results at a lab meeting the following week and Dr. Mason said, "wow I didn't think it was going to work." I'm glad it did, because I was able to advance my project now. To get to sequencing I have to walk across a few blocks to get to the building. I always look quite funny delivering my tiny pcr tubes through philly.

Me and my tubes :)

The sequence came back and I received a waveform, but this time without the aid of DSAP. However, because of my DSAP expertise I basically followed the same steps. The waveform verified that I did clone the correct gene, however it had 2 mutations and was incomplete in some areas. After looking at the amino acids I concluded the mutations were silent and wouldn't interfere. This was one part of the sequence with the mutation highlighted.

amino acids in the middle!

The next step of the project would to clone this insert into a specific vector and transduce bacteria, which I did during week 5! On my walk through campus on week 4 I saw the "love sign."

we love research!!!

During week 4 I became a pro at optimizing PCRs and cloning big genes that my PI didn't think I could.