Week One

Hi everyone!

I guess I should start out describing what exactly I am doing for my project. I am working with a team in a lab in the Molecular and Cellular Biology Department at the University of Arizona. What we are doing is screening potential drug candidates for their ability to counteract the toxic effects of TDP-43 expression in motor neurons. TDP-43 is a multifunctional protein that, among other things, is a telltale marker of ALS. ALS is a neurodegenerative condition in which the motor neurons degrade over time, and they cannot properly communicate with muscles. One of the projects that the lab is undertaking is screening different drugs for their effectiveness against TDP-43 and therefore, also ALS. This is achieved by feeding fruit flies various foods with different drugs in them.

We have to make sure that the fruit flies we harvest actually have the TDP-43 genes and mutation. The way we do this is a bit tricky. We put males with the TDP-43 gene in test tubes with females that have a different protein complex, called GAL4- D42. GAL4 connects to a region of the TDP-43 genes, UAS, and then enable the TDP-43 to be expressed and bind to the D42, which means that the offspring of these flies will express the characteristics of ALS. All of these proteins expressed in these flies are human proteins. In front of the TDP-43 gene on the sequence, there is a Green Florescent Protein (GFP) marker. This is a protein that, as the name implies, will exhibit a green florescent light when put under a ultraviolet light. This is a back-up check, to make sure that the flies actually were the result of a cross with a male with TDP-43 and a female with GAL4-D42. We are working with several different types of TDP-43, several different mutations that occur on that portion of the gene.

So that was a lot of science which pretty much boils down to having males flies with TDP-43, females flies with GAL4-D42, and baby flies with the characteristics of ALS (in this case, crumpled wings, impairing motor function.) These parent flies are put into a test tube to do their business, and after seven days, the parents are taken out. They have, at this point, laid eggs with lots of little baby flies. On day fourteen, we do a screen: we look to see if there are pupa and larvae, and if flies have already hatched, we see if they have straight wings of crumpled wings, and we counted a record the number of flies. If they have straight wings, they are counted as a "rescue", because they have recovered from the symptoms of ALS, crumbled wings. After all the flies are counted, the ones with straight wings are taken and put under a microscope, where we check to make sure they contain the GFP. If they do, they will glow, which is pretty cool. If the flies do not light up, it shows that they were not actually the result of a cross between a male with TDP-43 and a female with GAL4-D42, which means they didn't actually have the characteristics of ALS, which means they aren't a true 'rescue'. On day eighteen, we do the same as day fourteen, but we also count the number of pupa in the container, so we can gain some statistical understanding of how many rescues were in a tube. Three rescues out of thirteen pupa is a lot different than three rescues out of twenty seven pupa.

                                                       Glowing fruit flies! Super cool!

So other tasks involved in working towards this end product of rescued flies includes pulling males from the TDP-43 stock and virgin females from the D42 stock. The females have to be virgins because otherwise we don't know if the eggs are the result of a cross between a TDP-43 male or a D42 male. I learned how to identify the differences between male and female fruit flies (you have to look for a darkened abdomen as well as their genitalia) and if the females were virgins (virgins have a little black spot on their abdomen, and they also often have slightly crumpled wings because they have just hatched.) We look at the flies under a microscope, collect the flies we need, put them in tubes, label the tubes, and store them in a 25 °C incubator. The flies we don't need (the female TDP-43 flies and the male and non-virgin D42 flies) are thrown away into this huge erlenmeyer flask filled with vinegar and tons of dead fruit flies. It is just as nasty as it sounds, and I pray that I never accidentally knock over one of those and have to deal with the tidal wave of fruit fly carcasses and vinegar.

                                       Female on the left, male on the right. Lovely looking, eh?

We also "flip" the flies every several days. We have a constant rotation of flies, four rows worth, and when you flip them, it means you throw away the oldest flies, the ones nearest to death anyways, and then transfer the flies from the first row (the newest flies) into a new container, leaving behind the egss they left. The eggs will then hatch, and we can harvest those flies. I flipped for the first time today. The first few tubes I fliped resulted in some escaped flies, but by the end of it I had it more under control.

So that is mostly what I have been doing the last several days. A lot of looking at flies under a microscope. I also attended most of a lab meeting on Wednesday, which was a very interesting experience. A member of the lab was presenting on his work in the lab, which is eventually going to get published. He was talking about translation differences of G298S9 and human wildtype 2 TDP-43. These are just a mutation of the genes and the wildtype, or the version normally found in nature. I could understand most of the beginning of the presentation, but later as it became more statistics focus, I got a bit lost. Eventually it became a group-wide discussion as the head of the lab found a flaw in the reasoning. I thought it was really cool how everyone bounced different ideas back and forth and discussed what would and would not work.

So that had been my week. I hope everyone else has had a great week too.  I'm super excited for the upcoming months and really pleased with what I have learned so far.