While every day has a different to-do list, two tasks remain consistent: collections and dissections. Every morning, we check our boxes of fly vials for the ones we need to collect from. With tons of crosses going at the same time, it’s hard to keep track. To collect the flies, we dump the vials onto porous pads that emit carbon dioxide to put the flies to sleep. Then, we can look at them under a microscope to see if they have our desired phenotype, genotype, and age! This process allows us to have flies with everything we need in their genotype by crossing lines that already exist. For example, with this method we can create lines with a mutated spastin gene, but only in their neurons! We can also make lines to control for the technology that we’re using; if the technology was causing a phenotype, then that could mean our spastin mutation isn’t causing the results we see.
Aside from collections, we also dissect larva of interest. Because mutants with deletions in the spastin gene have bunched terminal boutons and tiny synapses, dissection is key for our lab to study different types of spastin mutants. Dissecting consists of placing a larva in a dish with pins in it. Using forceps, pins, a saline solution, a microscope, patience, and expensive tiny scissors, we pin the larva down and create “fillets”. We fix these fillets so that they remain stable while they go through several washes, solutions, and antibodies. Finally, we mount the fillets onto a slide so that we can look at them under the microscope with fluorescence. Recently, we learned how to “score” a slide, which means we count the number of terminal boutons on a particular muscle. Difficulties arise when boutons synapse on different planes of focus on the microscope, meaning they aren’t synapsing on the same muscle even though they seem to be in the same area. Despite having difficulties with scoring, I’m beginning to develop an eye for finding muscle 4 and counting the terminal boutons that we are interested in!
In addition to collections and dissections, our days consist of working out crosses on the white board, talking with Dr. Sherwood about the science, or going through papers. We also recently started a PCR project to determine if some lines actually have spastin deleted from their genome. Even though it’s been a struggle at times, Shibani and I have learned a lot and have developed our skills in collection, dissection, paper reading, scoring, washing, staining, and more!