A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The overall goal of the following experiment is to identify mitochondrial toxicity or beneficial effects of drugs using the model organism. C elegance. This is achieved by the in vivo quantification of a TP levels in transgenic sea elgan strains that express the firefly cruciferous enzyme fused to the green fluorescent protein.

The fusion gene confers two distinct properties to nematodes. The first is that when given the substrate Lucifer, the nematodes emit light in the visible range in proportion to their cellular A TP reserves. And the second is that when illuminated with the appropriate wavelength, they will exhibit strong green fluorescence.

Fluorescence is independent of the nematodes a TP levels and is proportional to their mass or numbers. Therefore, it can be used to normalize the bioluminescence measurements. During a typical assay, a population of developmentally synchronized worms is grown to the L four larval stage.

The nematodes are then treated with the test drug compounds for a period of time prior to the measurement of both fluorescence and bioluminescence. Results show potential drug toxicity. When bioluminescence is reduced, an enhanced signal indicates a hit for further testing for beneficial effects on mitochondrial function.

The main advantage of this technique over existing methods like drug screening on cultured cells, is that screening is being carried out in the physiological context of a whole live multicellular organism with close to 50%genetic ophthalmology to humans. And like humans, the elegance actually requires a mitochondrial oxidative phosphorylation to develop into adulthood and its mitochondria also share with mammalian mitochondria many characteristics in terms of their structure, their function, their bioenergetics Critical parameters within the protocol include the developmental stage at which the exposures initiated the length of exposure to the of the nematode, to the drug of interest and seeding of similar number of nematodes to each of the wells, and of course, avoidance of contamination On day one. Under sterile conditions transfer nematodes from one six centimeter nematode growth media plate in two milliliters of S complete medium into a flask with 30 grams per liter of e coli.

OP 50 in S complete medium, then incubate the flask with shaking on day four, bleach the culture of GR nematodes to harvest the eggs and to synchronize the worm population. Then incubate the eggs overnight in a glass flask containing 15 milliliters of S complete medium with shaking the next day, day five, the nematodes will be at the same stage of growth. To determine the number of hatched worms using a clean tip each time, transfer three 100 microliter aliquots of nematodes into separate micro tubes containing 900 microliters of medium, supplemented with 0.01%tween 20 pipetting up and down a few times to release any worms adhering to the tip.

Then count the nematodes in four separate 10 microliter droplets from each of the triplicate dilution tubes, and calculate the volume of the nematode suspension required to set up two 20 milliliter cultures with 10 nematodes per 10 microliters, decant approximate volumes into two pre weighed 15 milliliter conical tubes. Weigh the tubes to determine the actual volume dispensed. Then to adjust the target volumes vortex gently and remove excess volume.

Now, wash the nematodes in a total of 14 milliliters of fresh S, complete medium per tube and resus suspend the pellets in five milliliters of S complete medium with 30 grams per liter of equali. OP 50. Transfer the nematodes to conical glass flasks containing 15 milliliters of S complete with 30 grams per liter of equali.

OP 50 to a total volume of 20 milliliters per flask and record the time of feeding. Average the number of nematodes in nine 10 microliter droplets to confirm that the cultures are diluted to 10 nematodes per 10 microliters. Place the cultures in the incubator for 42 to 44 hours.

On day seven, pull the nematode cultures into a single flask with continuous gentle swirling. Then transfer three, four milliliter aliquots of nematodes into a single 60 milliliter sterile trough. Use an eight channel pipette to Eloqua, 25 microliters of suspended nematodes per well into 96.

Well black microtiter plates with flat, transparent bottoms. Then return the lids to the plates and set the plates aside. Each time two plates have been seeded, transfer another two, 2.5 milliliter aliquots of nematodes into the trough to replace the lost volume.

After seeding 13 to 14 plates with nematodes, add 74 microliters of S complete medium to each well and transfer the plates to a damp chamber and a shaking incubator until the drug administration, while the plates are shaking. Thaw 2 96 weld drug plates for testing. Then using a multi-channel pipette and changing the tips each time.

Transfer one microliter from the first column of the first drug plate to columns one to five of a nematode plate. Transfer another microliter from the second column of the drug plate to columns eight to 12 of the nematode plate. Then add one microliter of vehicle to columns six and seven of the nematode plate.

Use the remaining columns in the drug plate to treat the rest of the nematode plates in the same way, making sure to include two all vehicle treated plates as well. Then return all of the plates to the damp chambers in the shaking incubator for another 20 to 22 hours on day eight. To prepare a background control plate for fluorescence readings, combine the well contents from an all vehicle treated nematode plate and allow nematodes to settle for two to three minutes.

Then collect the bacteria containing snat and load 100 microliter samples into each well of a black microplate with a transparent bottom. Observe the plate under a microscope, noting the wells in which the nematodes can be seen to allow exclusion of these wells from the fluorescent background estimate. Then read the fluorescence finally to measure the bioluminescence for each plate.

In turn, dispense 50 microliters of freshly prepared luminescence buffer to each, well place the plate on a shaking platform and start the timer after three minutes. Read the luminescence at one second per measurement. In this first representative experiment, rot known a mitochondrial complex one inhibitor reduced the light output by the nematodes after both relatively short and longer exposures at a range of concentrations.

In this experiment, the herbicide paraquat was demonstrated to significantly decrease the bioluminescence GFP fluorescence and normalized bioluminescence of the C strain PE 2 54. Further, the bioluminescence decline was greater than that of the fluorescence, consistent with the known effects of decreased mitochondrial function and reduced a TP production elicited by the drug. This graph illustrates an example of a compound that enhances the nematode bioluminescence, the citric acid cycle intermediate oxaloacetate at a single concentration of eight millimolar.

Note that the experimental variability seen between these replicate runs is not unusual for Lucifer based experiments. Here, Firefly luciferase inhibitory compounds were observed to affect the in vitro lucifers activity at 25 micromolar and 100 micromolar with an almost complete attenuation of the activity by the DDD compound. 1, 4 3 4, although not directly comparable, a significant decline in the bioluminescence was observed after Lucifer inhibitory compound treatment in vivo as well.

Demonstrating that changes in bioluminescence may result from the action of Lucifer enzyme inhibitors rather than from changes in the in vivo A TP levels. While carrying out this procedure, it's important to consider that compounds can affect the activity of the firefly luciferase enzyme directly rather than affecting the worm's energy status. And therefore, sub heats can be validated using many techniques for assessing mitochondrial function, like, for example, determination of oxygen consumption, mitochondrial membrane potential determination of reactive oxygen species, or visualization of mitochondria in strains of sea elegance.

With GFP tag, mitochondria.