My project in the lab is to analyze the effect of the TREM 2 gene on macrophage polarization; the hypothesis being that TREM2 upregulates macrophage polarization and positively drives regeneration. The procedures I perform to accomplish the further analysis of TREM2 varies day by day. The entire process begins with the knockout of the TREM2 gene in mice, which we will contrast to wild type (normal) mice. To knockout the gene successfully involves a series of specific breeding amongst the mice which is carefully orchestrated through conscientious genotyping. After that step is accomplished, we induce an injury to the two mice to test the different levels of regeneration. We perform what is known as a Hind Limb Ischemia (HLI) surgery on the mice which involves ligating the femoral artery in the hind limb of the mice to prevent adequate blood flow. Subsequently, we collect the muscle tissue from this damaged area and dissociate the tissue into single cells. Those cells are then further analyzed and sorted by being run through a FACs sorting machine. FACs allow us to categorize specific cell populations based on phenotypes and to better understand the characteristics of a single cell population. By this method, we separate and collect the macrophages present in the sample. After collecting, we then isolate the RNA in order to better gauge which genes are being expressed and their relative abundance through qrt-PCR. We look for specific M1 and M2 markers in order to better understand the turnover of the macrophages and whether or not one is being expressed more or less as a consequence of the TREM2 knockout.
As an alternative method of analysis, we perform staining on the muscle tissue samples themselves. One particular stain, H&E, involves performing a cross section of the tissue and fixing the sample in solution in order to get a clear visual. Through this, we can clearly see which fibers in the samples are effectively regenerating and which are not. To further verify, we perform immunostaining, which involves the staining of a specific marker of regeneration (if there is increased staining, that means there is increased regeneration in the respective sample).
A smaller project we are currently working on involves culturing the cells we obtain from the muscle tissue samples into Bone Marrow Derived Macrophages (BMDM). We take BMDMs from each muscle tissue, the wildtype and the TREM2 KO, and induce the polarization of specific macrophages. We accomplish this induction by subjecting the cells to specific mediums such as IL-4/LPS that are known to cause polarization of specific macrophages. We then hope to perform a similar experiment as before in which we isolate the RNA and perform a qrt-PCR to see, once again, if the turnover of macrophages is affected in the different samples.
My experience thus far has been a blast! Thanks for reading!
