Colin King, Weeks 4-6, Making rats turn blue and other fun stuff

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 Miniprep or Midiprep doesn’t get you enough of the final DNA solution to work with. The first step simply requires taking your DNA you want to obtain and transforming it on bacterial plates, before picking colonies and growing them in overnight in a larger flask. However, every time I would do that, I would come in the next morning, and my flasks would be clear, indicating that nothing had grown and I had wasted 2 days doing the aforementioned procedure. After repeating this step 3 times, making sure I had done the process right each time, I came up with the same result, and had no idea why. I had no reason to believe it was the bacteria I had picked, since colonies were growing on my plates, indicating the transformation was successful. I had picked “Top10” a type of e-coli that grew faster and was the most commonly used type. Since I had nothing to lose h, I just picked the other type, “Stbl3”, for my fourth transformation and prayed to god it would work when I grew it in the large flask overnight. After doing some research, I realized that it was in fact Stbl3 that I should’ve been using all along,, as apparently it is more suited for taking a gRNA construct with multiple gRNAs, as mine was. I sighed in relief, finally confident that the next day I could go in complete the Maxiprep.

Even though I wasn’t allowed to observe the hydrodynamic tail vein injection of my genes into the mice (which is the process in which the gRNA and cas9 is injected through the tail), I was able to observe a normal tail vein injection for rats while he was instructing members of the next door lab. This was my first experience with live animals in any lab, and it was exciting to finally see. Since the tail vein injection this time was purely for instructive purposes, Frank only injected a saline solution with some blue dye. As he showed me (and actually let me feel out for myself) the location of the tail vein, he explained in great detail how the process of tail vein injection worked.

“I mean, you kind of just stick it in, and inject. Like obviously make the needle run parallel to the vein, then stick it in. The vein is closer to the surface of the skin than you think. And I guess practice?”

Frank performing the tail vein injection

He showed the member from the next door lab that he was instructing how to do it a couple times. The rats were sedated with some type of anesthetic, their tiny mouths pressed up to a little gas mask type of contraption, within seconds they fell limp, their tiny chests moving up and down just slightly as they took a breath. Frank made it look as easy as he explained it, within seconds the mouse turned blue, indicating the injection was successful. By far, that 6 second injection most fascinating thing I’ve seen yet at the lab. The rat was returned to a cage, and within seconds it was back to running around its cage, the only change in its appearance being its color.

However, the injection proved far more difficult for the person Frank was instructing. He attempted the injection 14 (yes, I counted) times before finally sticking it into the vein and injecting the saline solution. The mouse turned blue, but by that time, the tail itself was completely blue as well, from all the misplaced punctures into it with the needle and unsuccessful attempted injections.

Afterwards, Frank turned to me and told me he’d have some mice for me to “play with” sometime soon as well, as long as we kept it on the down low.

               Since it’s about a two week wait till we can do screening on the mice to see if the genes are knocked out. For the mean time I’m working on making gRNA constructs for Mercedes. It’s a bit disheartening to send off 14 tubes for sequencing (which is like 45 bucks) multiple times, and have each sequence result ending up as a failure when checked against the written construct on the computer, and going back to do yet another ligation or digest, but hopefully I can get these done and ready for injection soon.

               3 weeks to go!