In the Caldwell Lab, AKA, the Worm Shack, we study neurological disease mechanisms in sea elegance. And after a long day of fruitful experimentation, we reward ourselves with some recreational activity. Hi, I'm Adam Knight, undergraduate student from the Calwell Laboratory in the Department of Biological Sciences in the University of Alabama.
Today, myself and others will show how to perform a dopamine neuron degeneration assay in C Elegants. I'll be crossing a dopamine neurodegenerative worm strain with transgenic strains that overexpress candidate disease related genes and dopamine neurons. Hi, I'm Adam Harrington, a graduate student in the Caldwell Lab.
Today I'll be showing you how to set up the transgenic worms for dopamine neuron analysis. Hi, I'm Shu Amichi and I'm a graduate student. Here I'll be showing you how to assay enter your dopamine neurons of C elegance, which are called ceps and aides.
Generally the worms expressing only GFP in the dopamine neurons do not exhibit cell death. However, in presence of CLE, you will observe those and age dependent neurodegeneration Using this assay, a transgene could be co-expressed in the dopamine neurons to determine whether it can enhance or suppress CLE induced toxicity. So let's get started.
Before carrying out the dopamine neuron analysis, we must first cross the appropriate GaN strains to begin place eight mills expressing both alphas nucle and GFP under the control of the dopamine neuron specific promoter dat one onto mating plate. Now add two L four stage transgenic ca aphrodites which exhibit dopamine neuron specific expression of our candidate gene of interest, as well as body wall muscle cell expression of the DS red two fluorescent selectable marker. It's important to note that for each transgene construct we generate three stable transgenic lines.
So this genetic cross should be performed individually for each of the three separate stable lines created. After two days, remove and discard the males after an additional two to three days. Inspect the F1 generation.
There are several male progeny and the mating was successful. Next, individually transfer to new plates five from Aphrodite L four animals from each cross that exhibit both the GFP fluorescent marker inherited from the male parent and the DS red two fluorescent body wall muscle marker inherited from the hamadi parent. The transferred animals need to be at the L four stage to ensure that they have not yet M it.
With the male animals present on the plate, allow the animals to self fertilize for three to four days to produce F two progeny. Now clean out the F two progeny to do this. Transfer five to 10 animals that exhibit both cent markers to their own individual plates.
Then screen the F three generation for plates in which a hundred percent of the animals express GFP in the dopamine neurons and which some of the animals expressed the DS red two fluorescent marker and body wall muscle cells. These animals will be homozygous for the alpha synuclein and the GFP genes and will also be stably transmitting the newly created transgene. Once the screening is completed, we are ready to perform the dopaminergic neuron analysis.
To begin the neuron analysis, prepare a fresh plate of the F three animals identified in the previous step. Set up a plate for each of the three transgenic lines. In addition plate, the alpha-synuclein GFP alone strain as a control allow the worms to grow to adulthood.
Once the worms have reached adulthood, transfer 30 to 40 transgenic animals from each line onto a fresh plate. Allow them to lay eggs for five hours at 20 degrees Celsius. After five hours, remove and discard.
The adults allow the embryos to develop for three to four days at 20 degrees Celsius until they reach the L four stage. Next, pick about a hundred transgenic L four animals to a plate containing 0.04 milligrams per milliliter of five fluoro. Two deoxy uridine.
This nucleotide analog prevents development of the next generation via inhibition of DNA synthesis. Since the FDUR plates are light sensitive, protect them from the light by keeping the plates undercover and wrapped in full. Make aug gross pad for the worm analysis.
These are used fresh and are not allowed to dry out. Set out two microscope slides that have a piece of tape across them.Lengthwise. The tape serves as a spacer for equivalently thick pads.
Lay a third slide between them on the bench. They're positioned. Three abreast.
Place a drop of molten arus on the center side and quickly lay another slide on top of the agros perpendicular two and across all three slides. Repeat to make several additional pads as many as are needed for the number of samples to be analyzed. Once the auger pads are ready, place a six microliter drop of three millimolar, 11 mile saw and anesthetic on a 22 by 30 millimeter cover glass.
Pick 40 adult animals into the drop and then invert the cover glass onto the agros pad. Repeat for each of the other lines. Score 30 animals per line for the presence of each CEP and a D neuron.
Using a compound microscope with epi fluorescence in a filter cube that allows visualization of FITC or GFP, we'll use an endo G-F-P-H-Y-Q filter cube record data on a scoring sheet. Wild type animals retain complete GFP for lessons in all four CEP neurons and two A DE neurons. Individual animals exhibiting loss of D neurons are scored as known wild type or degenerating.
These neurons are recorded as LG or loss of GFP. Likewise, the extent of degeneration can be scored by counting the total neurons lost in each animal as well as the population repeats the analysis two more times. Using 30 more animals per line per trial.
The total number of worms exhibiting wild type dopamine neurons from the three rounds of analysis is averaged. Statistical analysis for neuroprotection is then performed using the student's T-test P value of less than 0.05. To compare control worms with strains that overexpress candidate genes in dopamine neurons carry out this analysis of various days following egg lane, the appropriate days of analysis of determined empirically.
For example, we have determined that 77%87%and 90%of alpha CLE plus GFP animals exhibit degenerated dopamine neurons at day six, seven, and 10 post development respectively. Therefore, if we predict that the transgene of interest might enhance neurodegeneration, we will analyze at day six. Likewise, transgene that might suppress neurodegeneration will be analyzed at days seven and 10.
We have just shown you how to perform a dopamine neurodegeneration assay in sea elegance. When conducting this procedure, it's important to remember to one, use appropriate marker for selection during genetic crosses. Two, use healthy males and L four HEDIS for the crosses.
And three, make sure you carefully and consistently score CEP and a DE neurons. This procedure will allow you to rapidly assay for genetic factor that may enhance or suppress alpha alpha CLE induced neurodegeneration. So that's it.
Thanks for watching and good luck with your experiments.
