Cryptococcus neoformans is a cryptic pathogenic yeast that is able to elude macrophages by replicating its chromosome set (among other cellular components) until it’s too big to be engulfed. The result are huge polyploidal cells, called titan cells, that are amazingly able to bud into haploid progeny. Just how this occurs is what my postdoc, Ci Fu, Dr., and I are currently researching.
The primary focus of this project are six cyclin genes that my postdoc chose as possible candidates associated with the C neoformans endoreplucation pathway. In the first week, I worked on creating gene deletion constructs (with a drug at the center) for the project, and by the end of the first week, I was able to create them all (shutout to PCR). In fact, by the middle of the second week, I had to make 8 copies of each gene which resulted in 48 PCR tubes.
Yes, I took pictures of my conquest….
Next we worked on cell culturing for two strains in order for us to do biolistic transformation. My postdoc has been working on a different strain, doing the same thing I am. But the absolute best part of the third week has been shooting a gun- a gene gun, that it is. This beautiful contraption allows me to create pressure in a chamber and literally shoot an aliquot of my gene (mixed with goldbeads) with helium.
…It’s not your average gun…but it does ‘fire’.
After at least 3 hours of recovery, I transferred each shot plate onto two drug plates, which resulted in 48 drug plates (each gene deletion construct was shot four times onto different plates). The idea here is that the genes constructs I made will have stuck to the goldbeads (using a washing process I completed beforehand). Once shot, it will kill the cells in the middle of the plate but transform the ones on the periphery. Since we don’t know which cells were transformed, we have to carry ALL the cells from the shot plate and select for them on a drug plate (I used water, a cell spreader, and a pipet to transfer).
The final part of the project is to see whether these drugs impact titan cell formation. So the third week and during this week, we have to determine conditions for titan cells. Last week, we found conditions for a larger version of my postdoc’s strain, but it wasn’t technically a titan. C neoformans becomes a titan in vivo when they fear being engulfed by macrophages in the lung of the affected individual. Getting these titan cells in vitro has rarely been done before. So my postdoc and I are essentially using the only protocol known so far for getting these titan cells (sent by another scientist). But, we weren’t exactly successful this past week, so this week we have to change the ingredients for incubation in 96 well plates- we limit nutrients so that these cells can hoard it into large vacuoles, a common characteristic of a titan cell. We plan on relying on the studies of a professor who started out in our lab- under my PI Joe Heitman, MD., PhD. – who is now revolutionizing in vivo studies of the pathogenic fungus: Kirsten Nielsen, PhD.
Dr Nielson and her team found this titan cell (in the red) in vivo. The macrophages are clear/gray, to the right and the normal C neoformans cells are in the blue.
Nonetheless, whether we get an actually solid answer from this project, I am so grateful for the opportunity to be here and learn so much. It’s been fun and exciting to learn new techniques and use incredible technology like a biolistic gun and a multichannel pipet. Everything has been amazing.