The overall goal of the following experiment is to assay toxicity of the beta amyloid peptide in an InVivo model system. The reproducibility and relative simplicity of this assay allows rapid testing of drugs, environmental conditions, or genetic modifications on beta amyloid toxicity. A key factor in developing this assay is the generation of transgenic cl egan's worms with muscle specific temperature inducible expression of the beta amyloid peptide.
This temperature sensitive expression is engineered by constructing a transgene that contains an abnormally long three prime untranslated region making transgene mRNA a target of the mRNA surveillance expression of this transgene is therefore poor in wild type worms with a functional mRNA surveillance system due to degradation of the transgene mRNA. If this transgene is introduced into a genetic background that contains a mutation in an essential component of the mRNA surveillance system, transgene expression will be significantly higher. If the genetic background contains a temperature sensitive mutation in the mRNA surveillance system, then propagation of this trenchgenic strain at the non permissive temperature will block trench gene mRNA degradation and result in high trench gene expression trenchgenic worms employing this system to regulate body wall muscle expression of the beta amyloid peptide temperature upshift from 16 degrees Celsius to 25 degrees Celsius, results in high accumulation of the beta amyloid peptide dysfunction of muscle cells and paralysis of the worms measurement of the time.
It takes a population of these trenchgenic worms to become paralyzed after temperature. Upshift is a sensitive assay for beta amyloid toxicity. This is achieved by making fresh nematode growth media plates for the paralysis assay, which helps minimize environmental variables that can influence paralysis rates.
As a second step, age synchronized populations of trenchgenic worms or initiated, this minimizes the effects of developmental stage on transgene expression. Next, synchronized third larval stage ones are upshift from 16 degrees Celsius to 25 degrees Celsius, increasing the stability of transgene mRNA and leading to the accumulation of the beta amyloid peptide. Measuring the time from upshift to paralysis for each worm captures the rate of muscle dysfunction, thereby assaying the toxic effects of beta amyloid peptide accumulation results are obtained that show the effect of specific treatments on toxicity of endogenously expressed beta amyloid peptide.
So the main advantage of our technique, as opposed to using transgenic mouse models to assay drug activity is that we can do ours much more rapidly and much less expensively. Visual demonstration of this method is critical. The scoring of L four worms in a paralysis assay is very difficult to learn.
So when you are scoring a paralyzed L four worm, what you wanna look at is the isolated halo movement of the head region of the worm. Once that's moving and the entire body of the worm is paralyzed, we would score that worm as being paralyzed. Paralysis essays are performed on plates of standard nematode growth media or NGM routinely used for CL egan's propagation and genetics.
To prepare plates autoclave, the NGM solution containing sodium chloride, agar, and peptide in an erlenmeyer flask, then add the appropriate amounts of the following sterile solutions that can be found in the accompanying written protocol. Aliquot 10 milliliters of liquid NGM into each 60 millimeter by 15 millimeter Petri dish. Allow the NGM to solidify overnight at room temperature.
If testing for the effects of compounds or extracts, aliquot the extract onto each plate and use a spreader to evenly distribute it over the surface of the plate. Allow the plates to dry overnight at room temperature. Volumes over one milliliter will require more time to dry.
Next spot each plate with 250 microliters of e coli strain OP 50 grown in LB media overnight to an optical density of 0.4 to 0.6, allow bacteria to dry overnight at room temperature. The age of the plate has a significant effect on the paralysis rate, and as such older plates tend to dry out. Plates should be poured within one week of the paralysis assay.
Ideally, you wanna use plates that have been poured three to four days before your synchronous egg lay plates used in any experiment should all come from the same batch altering the size of the lawn and or the bacteria will also alter the behavior of the worms Paralysis assays can be performed using alternative e coli strains. For example, HT one 15 is used in RNAi experiments. However, this may change the kinetics of the paralysis assay.
Therefore, you have to use internal controls that are appropriate Strain. CL 476 is most commonly used for assaying a beta induced paralysis. This strain and other transgenic models are available to academic researchers for a nominal fee from the Saint Ahadi Genetic Center.
To maximize the age synchrony of the test population, it is preferable. Start with a synchronized parental generation. A week before initiating the paralysis assay of the test population, use a platinum worm picker to transfer 20 to 30 grave adults onto several 10 centimeter NGM plates.
Spread with OP 50 for two hour egg lay at 16 degrees Celsius, which is the permissive temperature for CL 4 1 76. Remove the GR adults and allow the progeny to grow for seven days by which time they will be second day grave adults. Second day gravity.
Adults are used to prepare the age synchronous test populations for each experimental condition. The object is to generate triplicate plates containing 50 to 75 age synchronous worms using a platinum worm picker Transfer 10 to 12 of the day, two graver adults onto 60 millimeter NGM plates spotted with OP 50. Allow the worms to lay eggs at 16 degrees Celsius for two hours, then pick off the adults and allow eggs to hatch and grow as 16 degrees Celsius.
At this point, it is best to have someone who will not be scoring the plates to code them so that the score will be blind to experimental conditions. The stage of embryonic development when the eggs are laid, which is approximately the 26 cell stage for wild type worms under optimal conditions, is a function of adult age and nutrition. If the gravity adults used for the egg lay are older or starved later staged eggs will be laid and the synchronous population will not be achieved, thus affecting the kinetics of the paralysis assay.
It is essential that monogenic bacterial cultures such as e coli, OP 50 be used because bacterial contamination of any kind can severely affect this assay. Reproducibility is highest if your triplicate plates have a similar number of worms To induce the transgene and carry out the paralysis assay. Upshift plates to 25 degrees Celsius.
48 hours after the end of the synchronous egg lay worm should be at the third larval stage. Plates should be arranged without stacking in a 25 degree Celsius incubator to allow plates to reach this temperature. At the same time, begin scoring the paralysis of worms at 18 to 20 hours after the upshift has been initiated.
At this point, all worms in the population should have reached the fourth larval stage, continue scoring in two hour increments until all worms on each of the plates are paralyzed. For unknown reasons, paralysis is not even across the body length. The head region is the last part of the worm to cease moving.
Thus, worms that have recently initiated paralysis cannot translate across the plate, but can move their heads, thereby clearing bacteria around their anterior and leaving a halo of cleared bacteria. These worms will invariably become completely paralyzed and therefore worms with halos are characterized as paralyzed. Some worms will not have halos, but will also not show spontaneous movement.
These are tested by prodding them with the worm picker. If a prodded worm cannot undergo full body wave propagation upon prodding, it is also scored as paralyzed for efficient scoring. It is easiest to move the paralyzed worms to an unspotted sector of the plate so that they will not be unintentionally rescored the next scoring period.
This also allows miscategorized worms to demonstrate movement, allowing a scoring correction to generate a paralysis curve. At each time point, the fraction of worms on each plate that have not been paralyzed is converted to a percentage, and the average unparalleled percentage is plotted against time from the time that upshift was initiated. The resulting curve is formally analogous to a survival curve and thus can be analyzed using standard non-parametric survival statistics.
The upshift of the transgenic myo three A beta worms must be performed by mid L four stage in order for paralysis to be initiated. The Myo three promoter is developmentally regulated. Once the worms have reached late L four or adulthood, the expression level has been significantly decreased.
The expression of the transgene is blocked if the worms are starved, so it's very essential that the worms are well fed throughout the experiment. We have never observed a paralyzed worm to recover under any condition. After 12 to 16 hours of Upshift, CL 41 76 will become paralyzed, regardless of if you downshift it to 16 degrees in strain, CL 41 76 upregulation of the transgene expression that causes paralysis can occur at lower temperatures.
However, please note that the paralysis rate will be affected in its timing. The paralysis rate depends on the specific transgene strain used. We've constructed myo three A beta strains that have a paralysis rate faster and slower than the CL 41 76, and as such, you need to adjust your scoring window for the paralysis assay accordingly.
Shown here a plot of a paralysis curve for CL 4 1 76, both untreated and exposed to 6.27 millimolar caffeine. This treatment results in a highly statistically significant delay in paralysis of approximately four hours, which we interpret as indicative of suppression of a beta toxicity. In this model, this experiment assayed the effects of ol another compound found in coffee.
The plot is typical for treatments that do not impact a beta toxicity. Note that in both experiments, about half the untreated CL 4 1 76 populations are paralyzed at 24 hours and paralysis is complete by 28 hours. These are typical timeframes for paralysis of control.
CL 4 1 7 6 populations significant deviations from this timing are indicative of altered environmental conditions or spontaneous deletion of transgene copies. See the accompanying protocol for additional information about strain CL 4 1 76. While attempting this procedure, it important to keep in mind the age of the plates, the stage of the worm population, and the ability to score the paralysis rate of the worms.
After watching this video, you should have a good idea how to use this sea elgan system to carefully assay beta amyloid toxicity. You should be able to detect quite subtle effects on this toxicity of a variety of environmental or drug treatments. The variables we've talked about are important to pay attention to because this will enable you to do the assay very reproducibly and with a consistent paralysis kinetics.
This is actually quite important if you want to compare your results done in your lab at different times by different people, or comparing your results to other researchers using this model system.
