A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

The overall goal of the following experiment is to perform a chemical screen in zebra fish embryos using manual sample processing. This is achieved by first placing adult zebra fish in into mating chambers with a divider overnight, and then removing the divider the next morning to collect embryos. Next groups of zebrafish embryos are arrayed into 96 well plates, and then treated with a drug at the desired developmental stage.

Then embryos are allowed to develop fixed with 4%paraldehyde and assay to gauge the effects of small molecules on the developmental process of interest. The results show developmental changes such as the gain or loss of gene expression domains that can indicate alterations in sulfate or tissue morphogenesis caused by the small molecules that were interrogated. The implications of this technique extend toward the clinical treatment for a variety of diseases because of the ability to identify potentially therapeutic compounds.

Generally, individuals new to this technique may struggle because they need to acquire the dexterity necessary for working with embryos, such as transferring them between multi-well plates without damage. Demonstrating the procedure will be myself and Eric Donahue, an undergraduate researcher in the lab To set up zebra fish matings. Use mating chambers with dividers place adult male zebra fish on one side of the divider and adult female zebra fish on the other side.

For every 96 well plate of small molecules to be tested. Set up 25 mating tanks and leave them overnight. The next day, remove the chamber that separates each fish pair and after one hour, use a fine mesh strainer to collect embryos before placing them in Petri dishes containing E three medium with a transfer pipette.

Remove debris from the plates and under a stereoscope. Observe each clutch of embryos and remove any unfertilized eggs. Decant the E three medium and replace with fresh E three.

Then place each dish of embryos in a 28.5 degrees Celsius incubator for two hours. Check the plates again and discard any additional unfertilized eggs. Continue to incubate the embryos to the 40%eely stage.

Remove the chemical library plate from minus 80 degrees Celsius. Use aluminum foil to cover it and allow it to thaw at room temperature with an eight channel pipette. Add 99 microliters of E three to columns two to 12 of a 96 well plate.

Then add one microliter of the appropriate compounds to the wells in columns two to 11 here, designated as the experimental samples Pipette one microliter of DMSO into the control column here designed to be column 12. Use aluminum foil to cover the dilution plate and return the stock chemical library to the freezer. Next, after selecting a Petri dish containing embryos at the 40%epi stage, use a transfer pipette to carefully place around 10 embryos per well into columns two to 12 of a 96.

Well plate. Use a glass transfer pipette to remove excess E three from each well and expel into a liquid waste container. To chemically treat the embryos, transfer 100 microliters of each chemical dilution from the 96 well plate into the respective well of embryos to create a humid chamber with a paper towel and place it into a light sensitive container.

Large enough to hold a 96 well plate. Then use a ceiling mat to seal the plate. Place it in the light sensitive container and incubate it at 28.5 degrees Celsius for the desired length of time.

To remove the chemicals, add 300 microliters of E three to each row of drug treated wells. Then draw off the medium before using E three to wash the embryos three times to fix embryos, transfer them into four labeled 24 well plates placing the pipette in front of a dark background. After each well transfer to identify embryos left in the pipette and to prevent crossover, remove any dead embryos.

Draw off the E three so that the embryos are barely submerged in liquid. Then use a glass pipette to add two drops of 50 milligrams per milliliter pronase to each. Well incubate the embryos for five minutes before agitating the wells to cause the Corian to break apart.

After using E three to wash the embryos twice, draw off and discard the remaining E three. Add 4%P-F-A-P-B-S to each well and incubate the embryos overnight at four degrees Celsius. Then perform a screen of interest such as hole mount in situ hybridization, or wish as outlined in the text protocol to score the embryos from the chemical screen.

Transfer them to labeled 12 well plates before using a stereo microscope to observe them as outlined in this diagram. It is practical for a single person to perform a chemical genetic screen on approximately 600 compounds over the course of nine weeks of dedicated effort, and that time can be reduced to three weeks if performed by three people. Thus, the bench time of one or a few people can bypass the need for high resource demanding equipment like automated systems.

As shown here, zebra fish embryos were subjected to individual compounds from a bioactive library from 50%of peopley to 24 HPF, and then assay by wish, using a cocktail of three ribo probes to detect nephron segments. As an example, grading system, a compound that resulted in a dramatically enlarged proximal convoluted tubule or PCT will be categorized as class one, which represents the class associated with the most severe defect and the highest level of confidence in the phenotype observed. The compound would then be annotated as class one PCT.

Plus, this classification provides a simple and fast way to describe a small molecule of interest, including the severity of its effect on the organism, the region that is affected by the drug, and the manner in which the region is affected. While attempting this procedure, it's important to remember to stage your embryos at the correct developmental time point. After watching this video, you should have a good understanding of how to perform a manual chemical screen with zebrafish embryos.