Before an architect lays the foundation for a building, they draw a blueprint in which they make their plan for the building. As the architect of this project, I needed to create my own blueprint to guide me in figuring out the effects of Sirtuin-6 on CAVD.
For my project, we used aortic valves from mice. Since previous studies have found that SIRT6 levels decrease as age increases, we used both young mice (3 months old) and old mice (14+ months old). Within these groups, we used wild type mice as a control group and genetically mutated mice as our experimental group. The genetically mutated mice are mutated by Pax3-cre-recombinase. Cre recombinase is able to recombine different genes without the use of cofactors. We used the cre recombinase to mutate the SIRT6 gene by recombining, or “floxing”, the Pax3 gene in the middle of the SIRT6 sequence. The wild type mice are “Cre-” because the cre recombinase was not used. The genetically mutated mice are “Cre+” because the cre recombinase was used. If this paragraph was a little overwhelming, here is a diagram that might clear things up a bit:
Although the mice were insanely cute, we needed to sacrifice them for the sake of science. R.I.P. mice. Some were sacrificed as young mice and some were sacrificed as old mice. Because many of the mice with SIRT6 reductions had premature deaths, we had a lesser amount of old mice in the experimental group of our project. After the mice were sacrificed, we took cross-sections of their aortic valves. We froze and analyzed these cross-sections in three different ways: Alizarin Red stain, immunohistochemistry, and echocardiography. I will be going into detail about these analyzing techniques in my next few blogposts. See you then!