Bridgid, Entry #1, Everything is going Swimmingly at CHOP

After 2 weeks of waiting, my time in the lab has finally begun! Unfortunately, I could not start exactly when I was supposed due to large amount of clearances I needed to complete before being able to work at CHOP.  Although it was hard to wait everyday until 4pm when the mail came, only to realize that my authorization didn’t come that day, it only made last Friday feel even more like Christmas when I got my special blue piece of paper, certifying that I was clear and ready to go!

Despite not being able to work in the lab until this Monday, I had been given an article to read relating to the project that I would be doing this summer. When I arrived on Monday, I then presented the article and led the discussion for our lab’s Journal Club.  The Journal Club, kind of of like a book club, is something that my post doc created for a few of us in the lab to get together and discuses articles having to do with the work that each of us would be doing this summer. I am so thankful that thanks to this term, reading an article like this didn’t scare me as much as it did in March! I was nervous going into it, especially because how well I could explain this article to a group of people that specialize in this field would be my first impression, but I’m glad that I had to opportunity to do so. Just in that short hour I already felt like I learned so much!

My lab bench

            Then, my post doc Patrizia, who I will be working closely with, showed me around the lab and introduced me to everyone.  Along with the two of us on the zebra fish project, there is a college student, Abby, working in the lab this summer.  In addition to the zebra fish team (which in my opinion has the most fun) there is another post doc, Karen, who does MLL target therapy work, and James, the lab assistance to my PI, who works with the MLL translocations.  Shortly after I got to meet everyone, Patrizia, Karen, Abby, and I all went to a seminar presented to the entirely oncology department at CHOP.  The seminar focused on T-ALL and the ground breaking clinical trials that are currently taking place in attempts to find targeted therapies to cure the cancer.  Although some of it was a little over my head, being able to be exposed to that kind of research at such a high level was so cool and gave me lots of hope that with all of the new technology we have and are developing, we can make huge strides in terms of precision medicine to treat pediatric cancer

patients.  For the rest of the day, I took care of some clerical stuff, including getting my ID, and set up for the experiment that we would be doing on Tuesday.

            On Tuesday, my day started with a 1.5 hour mandatory research safety training meeting After learning every hazardous chemical known to man and memorizing the safety procedure for what too do if it spills or if it is taken into the body in any way, I finally headed up to the lab to begin my work! My project with Patrizia this summer focuses around the MLL mutant.  We will be testing to see if injecting one of the transcription factors, pu 1, down stream of the MLL gene, that normally goes down in quantity when the mutation occurs, we are able to rescue the colony of hematopoietic cells back to forming regular blood cells. In order to do this, we need to do an RNA extraction from the zebra fish embryos to eventually look at gene expression. Relating to this, I had two main projects for the week, RNA extraction and cDNA.  Because it was my first time performing the protocol and Patrizia wasn’t sure when I would be starting with the clearances, in addition to the fact that the embryos are extremely hard to work with and we only get a certain number per breeding pair, I did the same RNA extraction protocol along side of Patrizia, except I did it with REH (human leukemia cell line) cells, instead of the zebra fish.  I started by dividing the cells into two separate tubes, harvesting them by centrifugation and then using a PBS solution to make sure that the mixture was homogenous.  I then added two solutions, TRIzol LS Regent and chloroform, which allows the mixture to separate into 3 phases: a pink organic phase at the bottom containing proteins, a white interphase with DNA, and a transparent aqueous phase containing our RNA.  We then completed the RNA isolation procedure, finishing with a RNA precipitation to end up with an RNA pellet at the bottom of the tube.  Then, we used the Nanovue to measure the quality and quantity of the RNA that we extracted.  I was so excited to see that for my two samples, I had concentrations of 334 ug/ ml and 1088 ug/ml and that my purity ratios (260/280 ratio) was a 1.9/2.0.  We then ran my RNA on a gel to look at the concentration of the two types of RNA 18S and 28S that were concentrated in my solution. 

            My other project for the week was creating cDNA. Starting using 1 ug of the RNA that we extracted earlier in the week (in my case the REH cell line and Patrizia’s case the zebra fish embryo RNA), we added water until we reached a final volume of 10 ul.  Then we added random primers, which anneal throughout the RNA molecule and give a good representation of the entire RNA molecule, and the dNTPs.  After rounds of PCR, then we added buffer, DTT (denaturing agent), and RNase OUT.  Again after a round of PCR, we added the reverse transcriptase enzyme (superscript II).  Finally we added RN-ase H, which breaks down the RNA so that we are left with only the DNA. 

            Since both of those projects were moving along nicely, Patrizia also had me start on DNA extraction from the zebra fish embryos.  Because the embryos have to be incubated over night, we prepared the embryos on Thursday to sit overnight and then when I came in on Friday we did the extraction.  The extraction mainly consisted using special tubes with a membrane to store the DNA on top and in the membrane, along with 3 to 4 washes with buffer and ethanol and spinning in the centrifuge.  Once we had out DNA, we ran PCR, which we will analyze on Monday.  Then using the PCR, this coming week, we are genotyping 160 embryos to look for the mutants.  On Friday afternoon, we had a lab meeting with my PI, where each of the summer students presented on what they had done so far this summer.  I was nervous since I had only been there for a week, but getting up and being able to explain what I have done in a very short amount of time to my lab was actually pretty cool (and great public speaking practice)!

            In addition to working with Patrizia, in my down time when Patrizia has post-doc meetings or is working her in situs, I have gotten to work with the college student Abby.  Although she is working with the zebra fish, she is working with a transgenic line, in which she is using a transgene in a vector inserted into the zebra fish to try and express the MLL-GAS7 mutation in the fish and mark it by fluorescent to prove that it is a gene that causes leukemia in zebra fish, along with humans.  I was able to work with her on her imaging of the zebra fish at 33 hpf (hours post fertilization). Through this imaging we found that the fish with MLL-GAS7 had a significant amount of more red fluorescence in the hematopoietic cells than the control.  This is ground breaking because now this is possibly a way to create

Zebrafish MLL-GAS7 imaging 

leukemia in a fish and allows the fish to become a model for MLL in identifying how the cancer occurs.

            Overall, my first week was awesome! Everyone is so friendly and knowledge that I feel like I have already learned so much! I can’t wait to see what the rest of the summer has in store!!