Crosses
ALS Crosses
We wanted to use the UAS-GAL4 mechanism to generate flies that express the GR80 gene in glial cells(repo). Our cross was:
We needed UAS-GR80/repoGAL4 flies for our testing. However our crosses didn't yield any flies of this genotype.
Methods
Virgin Collection
First, we had to collect virgin female fruit flies with the repoGAL4 / TM3serGFP genotype. Then we could cross these flies with male flies of the UAS-GR80 / CyO genotype (experimental) and UAS-GRcontrol genotype (control).
To identify virgin females, we separated the flies by sex, getting rid of the males, and then looked for a dark spot on the abdomen for the females. Virgin collection varied week to week, making it difficult to set up crosses regularly. So some weeks due to low virgin numbers, we only set up an experimental cross and not a control cross.
Setting Up the Crosses
Ideally, every time we would separate the virgins into two groups (experimental & control) and add males of the correlating genotype. The number of males added was more than the amount of females that were in the vial. Often, our numbers for each cross were around 2-5 virgin females and 3-6 males.
Progeny
After waiting about 10 days or more, we looked at the progeny from the crosses. From these flies we wanted to collect those with the repoGAL4 / UAS-GR80 genotype (no curly wings, no notched wings) for the experimental cross and those with the repoGAL4 / UAS-GRcontrol genotype (no notched wings) for the control cross. We wanted to use these flies as our test flies, because they express both repoGAL4 (for the glial cells) and UAS-GR80 or UAS-GRcontrol (for ALS).
Results
When we went to look at the progeny from the experimental crosses, we found that there were zero of the flies that we wanted to collect (repoGAL4 / UAS-GR80).
| UAS-GR80 | CyO----------------------------------------------------------------------- repoGAL4 | 0 flies | 15 flies-----------------------------------------------------------------------TM3serGFP | 12 flies | 10 flies
This suggests that these flies are dying. We then did some brainstorming as to why they are dying and how we could test this.
Glia are dysfunctional, leading to neuronal dysfunction OR death -> use immunohistochemistry to look at activity-dependent transcription factors (immediate early genes) levels
A specific subset of glia is causing fly death -> use specific GAL4 subsets and cross with GR80 and evaluate fly survival
A subset of glia is dead -> cross with UAS-GFP +/- UAS-GR80 & if GR80 kills, GFP will disappear
When are the flies dying? -> cross UAS-GR80 with ScO / CyO-GFP to get UAS-Gr80 / CyO-GFP which will enable us to use GFP to evaluate the phenotypes in each life stage
We have started testing the last point.
Up Next
Since our target flies are dying we want to figure out what's causing it and also when they are dying. For this we'll be using the cross,
UAS-GR80 Sco
---------------- x --------------
CyO CyO-GFP
And then we will take out those expressing Sco (those with missing bristles) to get UAS-GR80/CyO-GFP flies.
We'll collect UAS-GR80/CyO-GFP flies from this cross and then we'll cross them with repoGAL4/TM3SerGFP. If we then put the flies under UV light, we can tell which flies have the GFP marker. Those without the GFP marker will be the repoGAL4/UAS-GR80 flies. But the cool thing about this is we can tell which flies have the GFP marker in various fly stages. So we can use this information to help determine when the flies are dying.