Colin King, Weeks 7 and 8, Mouse Livers and Cell Lines

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, I enjoy the hands on application and practice of the lab work much more, and luckily this past week, Frank was able to allow me to practice some tail vein injections on my own, in addition to allowing me to observe the harvesting of mouse livers.
The mice were much smaller than the rats I had previously observed, and Frank had brought over around 10 of them so Nika, Sebastian, and I could all practice the tail vein injection. The mice were first placed under a large heat lamp in order to make the veins "pop" more. After a few minutes, they were clearly visible as large black lines running down the length of the tails. Hydrodynamic tail vein injection describes an injection with a solution of about 10 percent of the mouse's body weight, so they were first weighed. However, unlike the rats, the mice weren't given any anesthetic, they were simply grabbed by the tail and put onto the weigh boat. Frank explained that mice, when put in a new environment elevated slightly or with some type of  physical border, wouldn't leave that area.

Mice under the heat lamp. 

Next, the mouse was placed in a  restraining device, which was pretty much a box that could open at one end and had a hole at the other end large enough for the tail to poke through. After making sure the mouse wouldn't move around, it was finally time for me to start. I prepared the syringe and took a deep breath. Gripping the tail in my left hand and curving it slightly over my finger to expose the point in the vein I wanted to target, I tried to tell my right hand not to shake as I approached the tail with my syringe. I wavered around the tail for a few seconds before attempting to just barely nick the surface and insert the syringe, and realizing I had missed right after making contact. Withdrawing, I exhaled sharply and tried again just a hair closer up the tail, and found that the tip of the syringe slid easily in, meaning I had successfully entered the vein. After this, it was imperative to get the full volume of solution into the vein within a few seconds, and to not move at all, lest you puncture the vein or accidentally slip out of it. About half way through though, I realized I had done just that, and the tail started to puff up as the clear solution made its way into somewhere it clearly wasn't supposed to go.
Unfortunately, after one injection, the mice had to undergo cervical dislocation, which Frank introduced to me by saying it was what happened to the dog in I am Legend.

Sebastian also instructed me on how to take care of cell cultures, which was a new experience completely, as it required a level of organization and cleanliness like never before, or as he described it, “you have to be really, really, anal about everything”. Taking care of cell cultures involves splitting them once they reach a certain level of confluence (basically, how much they’ve grown on the plate; 100% confluence would mean they completely cover the plate), changing the cell media, and observing them everyday. Any work with them is done in a separate clean room, under a vacuum hood, which is first sprayed vigorously with ethanol and left under UV light to kill any bacteria.

Hopefully, the gRNA construct I made and Frank injected into mice last week will work, and we can soon harvest the livers and determine whether or not I successfully knocked out the gene. If there is time, I hope I can get some work with a western blot or ELISA, which are other methods of screening and observing the results from the construct and cas9.

With less than two weeks left, I just want to soak up as much as I can of the Bissig Lab.