Axons Weeks 7-10: Almost there

As my time in Dr. Buchser’s lab this summer came to a close, there was a mad dash for more experiments and more data. My fellow lab member, Karen, got the virus ready to go just in time for us to do some in vitro experiments with zebra finch neurons. While we’re still hesitant to call it a success (what is science without some healthy skepticism?), the initial results look very promising.

First, I’ll give a little more background on our method for knocking out the SARM gene.  It’s a system called CRISPR/Cas9 and its gene editing capability for cell culture was discovered in 2012. There’s a pretty cool Radiolab episode about it here if you’re interested! The point is that you need a virus to deliver the Cas9 protein as well as other viruses to deliver guide RNA, which targets Cas9 to the gene you want to manipulate.

For the experiment, we plated dissociated retinal neurons from zebra finches and gave them time to grow axons. Then we tested four different conditions: no virus, Cas9 virus only, guide RNA virus only, and both Cas9 and guide RNA viruses. After allowing ample time for infection and SARM knockout, we used taxol to induce degeneration and compare the different virus conditions. I then took some confocal images of the neurons and looked for fragmented axons.

We need to do more replicates, but the data for this experiment looked great! The neurons that got taxol and both viruses appeared to be less degenerated than the neurons that got taxol but one or no viruses. Sequencing the genomes of the neurons will give us more data, but for now we’re hopeful that our viruses are working and that we knocked out SARM, giving the observed protective effect against degeneration.

As you can imagine, I was excited that we got some good data after all of our work this summer. I’m definitely looking forward to continuing this project during the school year.

Comments

  1. The CRISPR/Cas9 method is absolutely amazing, and it sounds like you’re using it for some interesting stuff. I’m confused though, is the guide RNA a sequence that you specifically dictate to match to the area of the DNA you’re working with?

  2. Yep! From what I understand, they target Cas9 to the SARM gene specifically.