Peddie EXP 2017

My final days at the Bissig lab have turned out to be the most eventful yet. In the past days, I've harvested the livers from the mice injected with my gRNA constructs, harvested the DNA from those livers, and analyzed that DNA. In addition, I learned to split live cell cultures and infect human cells with viruses, and continued cloning gRNA constructs for other lab members.  Of the above, the dissection and harvesting of the livers was by far the most interesting experience. My constructs targeting the Agt and Ddc genes were injected into several mice each, and since it had only been a little less than two weeks since the injection, we couldn't preform many of the assays to determine what the effects of cutting into the genome had been. However, what we could do was harvest the livers of just one of the mice, and determine if my gRNA had been successful in leading the cas9 to cut in the right place. Essentially, whether or not what I had spent about 8 of the last nine wee...

After having injected the gRNA constructs I made with the cas9 to see if I could successfully knock out my target genes, I've mainly been focusing on helping out with other projects around the lab, mainly with Mercedes' project in synthesizing some other constructs. In addition, Collin has been instructing me on how to actually figure out how to pick genes to target in various metabolic pathways, and how to make up the gRNA construct on the computer. Since my gRNAs are for the murine (mouse) model, he has assigned me to creating one for a human model (which, when synthesized, will go into humanized mice , not actual humans).  This mostly involves research into the genes and metabolic pathways of interest and figuring out which exons are most important, and through using a few fancy programs on the lab computer, how to alter them in a way that can generate a frame shift mutation to knockout the gene. Although the computer work has still been interesting and no doubt important,...

Finally, after the arduous process of preforming a Maxiprep, I’ve successfully created my gRNA construct. Amazing, the process of 6 weeks of straight work, all for two little tubes of 300 microliters of what looks identical to water. The constructs were injected into two mice down in the mouse lab, which apparently I’m not even authorized to set foot in, to my disappointment. Dr. Pankowicz and Collin both assured me, though, that the injection was successful and within a couple weeks, we should have some mouse livers to harvest, and truly see if the construct was successful and if the genes of interest were knocked out.  Maxiprep materials Some appetizing cell debris after the DNA has been removed (step 5 of the maxiprep) The Maxiprep itself was especially annoying, only because I messed up the first step for close to a week straight. A Maxiprep describes a process where you can amplify and get large volumes of plasmid DNA in for processes like injections, when a Minipre...

Colin King, Week #3, The Smell of Water As I approach the end of my third week at the Bissig Lab, I’m finally beginning to achieve a sense of familiarity and comfort with my surroundings and the people who work around me. Dr. Pankowicz has returned, and though I usually don’t see him till he arrives after lunch (but apparently stays till 2 am), he has been extremely friendly and done a great deal to help me understand things however I have trouble or ask him questions (which is pretty often). Since my last entry, I have successfully synthesized the two gRNAs (guide RNAs) individually for each of the genes I was assigned to ( Agt  which is responsible for the creation of angiotensin II and  DDC  which regulates dopamine uptake). Since we want the Cas9 enzyme to effectively cut out one portion of the gene, from one exon to another, and each gRNA targets one exon in the gene, I now have arrived at the step of “Daisy Chaining” the two gRNAs together in one vector, so...

Having already familiarized myself with the procedure that I would be preforming for the beginning of my time at the Bissig Lab, I thought I would be quite prepared to start work and that everything would go smoothly. To say that this was true for my first week would undoubtedly be an overstatement, but in my opinion, it hasn't been to rough settling in. The people at the Bissig Lab have made this transition much easier. My PI, Dr. Bissig, will unfortunately be leaving in a few days for a 2 month vacation, but he's certainly sacrificed a good amount of the remaining time he does have in order to show me around and bring me up to speed on the lab's progress. Dr. Legras and Dr. Pankowicz (who was only Mr. Pankowicz till about 4 days before I arrived) have also been extremely helpful. Apparently, Dr. Pankowicz will be acting as my primary "mentor" for the majority of the summer, and I'm pretty glad to have someone as laid back and easy to talk to as him.  I...