Methods
Coding
To identify genes of interest, we created code in python that merged the intersection between several data sets created by various researchers. We first used Datacamp modules to learn some Python commands, and used google CoLab to help us merge datasets.
Merging Data Sets
Our first step was to merge two data's sets, one containing genes coding for plasma membrane located proteins and one containing genes coding for secreted proteins. Our primary interest in ALS is genes that are related to signaling pathways This code allows us to ensure that all of the genes in our final dataset are genes involved in signaling. To merge the two data sets, we first identified the overlapping columns "Gene" and "Proteins Class". Next, we used the Pandas commands "pd.merge" to merge the two data sets.
Adding the ALS Data Set
Our next step was to intersect our protein coding gene data frame with our dataset of ALS associated genes. The data that we used was from table two in the D'Ericha neuronal expression ALS dataset. We converted the data set from a pdf to a csv file and the performed an intersection with our previous data frame using the pd.merge "inner join" command.
Finally, we exported the merged data set into excel and manually filtered it to contain only genes that had significant change in expression in ALS (>1.5 Log fold change). We then had two separate csv files, one with genes upregulated in ALS, and one with down regulated genes in ALS. Pictured above is the spreadsheet with genes that are upregulated in ALS that we used to choose our genes of interest, highlighted in blue.
Our Genes
VAMP7/8
COMP
TIMP
Fly Collection
As drosophila are used for testing, the identification of sex is crucial to highlight sex-specific results. By using small amounts of CO2 to stun the flies, it prompts the researcher a chance to carefully examine the flies.
To identify a male:
Darker tip on the abdomen as seen in the picture
Have darker bristles, otherwise known as sex combs on the front of their legs
To identify a female:
Paler abdomen as seen in the picture on the right
(Mestetskiy et al., 2022)
(Brain VIP, Team 1, Spring 2023)
Identifying Virgins
The collection of virgins are detrimental to lab as it provides the opportunity to intentionally manipulate our progeny's genetics to our desire.
To identify a virgin:
Look for indications that a fly has newly hatched, this is usually a brown larvae-type of case
Virgin females are seen to be very opaque and white; being the prime identifier
Crosses
TIMP
(+/+ background)
Over-expression of this gene is expected to lead to hindered motor function in Wild type background flies.
Crosses of this type can be setup normally.
COMP & VAMP7/8
(ALS background)
Less expression of this gene is expected to lead to better motor function in ALS background flies.
For crosses of this type a more composite process is needed:
3 elements needed:
UAS-X (2nd chromosome)
alrmGAL4 (3rd chromosome)
UAS-TDP43 (3rd chromosome)
We may not want to put alrmGAL4 and TDP43 in a stock together, as they are expressing the disease gene constantly. So combining elements on chromosomes 2 and 3 immediately is not wise.
Instead we should cross UAS-X with UAS-TDP43. But, we need to be able to follow the chromosomes phenotypically. To do this, we can use a Double Balancer.
Experimental Designs
Behavior Testing
To measure ability of drosophila in performing geotaxis, a behavior test was employed. Crosses of flies are tested every week to serve as a rudimentary measure of ALS development, as ALS impairs ability for the flies to climb the testing cylinder. (Madabattula et. al.)
Behavior Testing Steps
Organize a fly vial of about 20 flies (preferably) from a cross, ensure their birthdate is present on the vial for organization. Document the information of flies and the date of the testing on a piece of paper to be shown on camera.
Ready a piece of parafilm to cover the top of the graduated cylinder, then transfer the flies from the vial to the cylinder.
Cover the graduated cylinder with the parafilm, also ensure the graduated cylinder has a soft base (folded paper towels).
Set-up the camera and focus on the yellow area of the cylinder, begin recording.
Show the piece of paper set up earlier to the camera briefly, then knock the graduated cylinder base against the soft base several times, moving all the flies to the bottom.
Record the number of flies that cross the 110mL yellow line every 10 seconds, up until the test has reached the 2-minute mark.
Upload video to BOX before the end of lab.