Colin King, Week 8 and Week 9

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 weeks doing was successful or not.

The first step was obtaining the livers from the mice, and Collin took advantage of the opportunity to show me a full dissection of the mouse, showing me where each organ was in the body and how to properly extract it. The liver was a large reddish brown mass with multiple lobes, and luckly for me, probably the easiest to remove. After slicing off a few pieces and flash freezing them, the next day we were able to harvest the DNA in the hepatocytes by dissolving a smaller piece of the liver and extracting the DNA through a purification process.

After running PCR to amplify the DNA we extracted from the liver, it was easy as running it on a gel to simply see whether or not I had successfully cut out the right part of the gene (or made any cut at all). If I had cut successfully, I would see two bands, one at 1500 kb (the uncut wildtype DNA, since only 30% of cells should have been transfected with my gRNA construct and had their DNA cut), and a band at about 700 kb. After waiting the painstakingly long hour for the gel to run, I finally walked over to the UV light and imaging area to see whether or not my entire project had amounted to anything. When the computer finally finished processing and displayed an image of the gel, I sighed in disappointment. There was one, dark band at around the 1500 kb mark for the Agt gene, and nothing at all for the Ddc gene. I had failed. I didn’t quite know what to say, but Collin fidgeted with the computer a little, changing the exposure, and near the 700 kb mark, the faintest band appeared in the well for the Agt insert. It was barely visible, but it was definitely there.

It was relieving, to finally have some validation of the work I had most of my summer on. The last day, I transferred over all of the cloning work I was in the middle of to Collin and ran some more PCR for Sebastian. Afterwards, we went out for drinks at a nearby cocktail bar to celebrate my last day.

This experience was one unlike anything I’ve ever had before, and I’m grateful to Dr. Bissig for trusting me with his research in his absence, and to everyone else in the lab for teaching me and guiding me through pretty much every step of the way. Hopefully, Dr. Bissig and Dr. Pankowicz will be willing to have me assist them again in the futue.