ALS is a progressive, chronic, irreversible disease. Multiple gene sets are found to be involved in the expression of ALS, each with varying degrees of toxicity, as evinced in the survival rates and locomotor performances of each fly group and stock.

 J.C. Wang, G. Ramaswami, D.H. Geschwind (2021) found and classified different modules with gene sets related in function. All of our genes were selected from the SC. M4. module, defined by Wang et alia.

This is a gene that codes for Deubiquitinizing Enzyme A, which breaks down ubiquitin proteins in the cell (FlyBase). Although it is currently unknown as to how it is implicated in ALS, we hypothesize that it is downregulated in ALS, as ubiquitin-proteasomes degrade proteins. Additionally, downregulation could make chromatin more easily accessible, which would make more transcription of DNA possible. This could cause protein aggregation, either directly or through epigenetic factors.

To successfully make a cross, it is vital that you are able to track the genetics of the flies you are crossing into their progeny. As mentioned in the introduction, flies have three main chromosomes. Balancer genes are used to maintain mutations and to more easily track traits. The main balancers we used in our crosses were Curly (CyO), Serrated (Ser), Scutoid (ScO), TM6bTbSb, and Lyra (Ly). It's important that you use a virgin female to ensure that you are able to properly track the genetic line.

Creating a Double-Balancer

Some of the crosses that we needed to make had our genes on the third chromosome. Due to the placement of the UAS/RNAi genes and the ALS genes, we needed to create a double balancer stock to cross our genes with to ensure they had all of the traits necessary. To start, we crossed a virgin female with the genotype +/+ ; ScO/CyO ; MKRSTM6b,SbTb with a male with the genotype +/y ; UAS-TDP43/UAS-TDP43 ; +/+. Additionally, we crossed a virgin female with the genotype +/+ ; +/+ ; alrmGAL4/alrmGAL4 with a male with the genotype +/y ; ScO/CyO ; MKRS/TM6b,SbTb. The progeny kept with both of these crosses were curly-winged that hatched from tubby pupae cases. To verify that the flies were hatching from tubby pupae cases, the cases were looked at before they hatched and separated based on whether they had the wild-type phenotype or the Tubby phenotype.

They were then crossed with flies with a genotype of +/+; IF/CyO ; Ly/TM6bTbSb. We picked against flies with the IF genotype and for flies with Ly, CyO, and TM6bTbSb. Unfortunately, we were unable to find any flies with all of the characteristics of a double balancer, so this will have to be restarted for next semester.

In the beginning of class, we were following the protocol set up by Madabattula et. al. We followed these steps:

1. Collect 20 flies of a given genotype

2. Transfer them into a 250mL graduated cylinder

3. Seal the top of the graduated cylinder with parafilm

4. Set up a video camera focused on the 190mL line

5. Press the record button on the camera

6. Tap the graduated cylinder against a padded surface in an unpredictable manner six times

7. Place in the marked area

8. Record the climbing behavior for 2 minutes

9. Upload the video to a computer

10. To analyze the video, count how many flies crossed the 190mL line every 10 seconds. If a fly drops back down below the 190mL line, it's marked as -1 fly

As we worked with this, though, we realized that the majority of our wild-type flies were not crossing the 190mL line. This was concerning in terms of our data, as flies with ALS genes are worse at locomotion. Due to this, we decided to alter the protocol so that the line that flies had to be crossed in order to be counted was at the 110mL line instead of the 190mL line. Additionally, we placed a piece of cardboard behind the beaker to make it easier to see the flies as they climbed. We were also not always able to use 20 flies, as some of them would die as they got older. Finally, we started adding a labeling section at the beginning of the video that stated the number of flies, their date of birth, and their genotype. Unfortunately, this last change was not implemented until about halfway through the semester, when the majority of our work was focused on our individual genes rather than group data. This meant that we didn't have a very solid stock of analyzed videos that we were able to use to find the percent that crossed the line, rendering them (mostly) useless in the long run.