Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

The overall goal of this procedure is to comprehensively assess germline chemical toxicity using the nematode C No Haitis elgan. This is accomplished by first synchronizing worms by adding a diluted bleach solution to a mixed stage worm population of the C Elgan PXOL one GFP strain. This step generates a large number of embryos protected from the bleach by their eggshell.

The second step is to generate a synchronized L one larvae population by letting the embryos hatch in liquid without food. Next, the worms grow to their fourth larval stage or L four, at which point they are exposed to the chemicals of interest in liquid for 65 hours. The final step is the visualization through fluorescence microscopy of expression of the GFP reporters in embryos inside the uterus of the worms.

Ultimately, the proportion of GFP positive embryos compared to the DMSO control reflects the antigenicity of the compounds tested. The main advantage of this technique over existing methods, such as Rodin models or cell culture assays, is that we can assess the genicity and germline toxicity of chemicals in a whole animal model while at the same time performing a high throughput assay performing the procedure. Today will be the postdoctoral fellow Dr.Daniella Perotti.

The graphic worms for this procedure were previously prepared as described in the accompanying protocol text, collect the GR worms from the nematode growth medium or NGM plates by washing them with one to two milliliters of M nine and transferring them to a 15 milliliter conical tube. Let the GR worm sediment to the bottom of the 15 milliliter conical tube for approximately five to 10 minutes. Next, remove the supernatant without disturbing the worm pellet.

Transfer the worm pellet to two to four micro centrifuge tubes and add one milliliter of diluted bleach solution to each tube. Incubate for two to three minutes at room temperature. Monitor the progress of the reaction under the stereo microscope to confirm all the worms are dead.

Do not bleach longer than five minutes as the embryos could die. Collect the worms by centrifusion for one minute. At 3000 RPM.

Remove the supernat and add one milliliter of sterile M nine to neutralize the reaction. Make sure all materials coming in contact with the worms after the bleach treatment are sterile. Wash the worms twice with M nine media.

Remove the supernatant and add M nine to 100 to 200 microliters. Using a glass pasture pipette, add a drop of the worm M nine mix to clear NGM plates and incubate them for at least 24 hours at 20 degrees Celsius to allow the embryos to hatch without food. The larvas growth will be halted at the L one stage Two synchronization steps.

Bleaching and L word starvation are being used because it is crucial to achieve a highly synchronous warm population to maximize the number of L four larvae that will be used for the exposure. One day after the establishment of a synchronized L one larvae population, collect the L one larvae from the clear NGM plates. Wash the plates with one to two milliliters of M nine and transfer the L one larvae to a 15 milliliter conical tube.

Let the dead adult worms sediment to the bottom of the 15 milliliter conical tube for approximately five minutes. Collect the supernatant where the L one worms are in a new 15 milliliter conical tube. Precipitate the worms by centrifusion for two minutes at 2, 600 RPM.

Remove the supernatant leaving approximately 500 microliters. Determine the concentration of worms in the M nine solution by counting the number of worms in 10 microliter drops. Using a stereo microscope count at least five drops.

Adjust the concentration of the worms to 20 to 25 worms per microliter in M nine, and then add 50 microliters of the worm M nine mix to NGM plates. Seated with OP 50, feeding bacteria incubate for 65 hours at 15 degrees Celsius to allow the L one synchronized population to grow until they reach the L four stage. This screen utilizes a CL against strain that contains a fluorescent transcriptional reporter to detect germline disruption and induction of embryonic aneuploidy.

Collect the L four larvae from the clear NGM plates by washing the plates with one to two milliliters of M nine and transferring them to a 15 milliliter conical tube. Let the L four worm sediment to the bottom of the 15 milliliter conical tube for approximately five to 10 minutes. Then remove the supernatant without disturbing the worm pellet.

Add three to five milliliters of M nine and determine the concentration of worms in the M nine solution by counting the number of worms in 10 microliter drops. Using a stereo microscope count at least five drops for each sample. Resuspend the worms at a concentration of one worms per milliliter in M nine.

Dilute the previously prepared OP 50 bacteria tenfold with M nine. Make sure the resuspended bacteria reach room temperature because lower temperatures affect worm development using a multi-channel pipette first, add 100 microliters of the worm M nine mix to each well of a two milliliter deep round bottom 96 well plate. Next, add 400 microliters of the diluted OP 50 bacteria to each well at the end.

Each well will have 100 worms in 500 microliters. Add 0.5 microliters of the test chemical to the desired wells to achieve a final concentration of 100 micromolar. A concentration commonly used in chemical screens in CL elgan expose ethanol or DMSO solvent controls at a final concentration of 0.1%as a positive control.

Use 100 micromolar no cosol. Seal the plate using adhesive film. Be sure to seal the plate well to prevent cross-contamination between wells.

Wrap the plate with aluminum foil. Transfer the plate to a shaker of the appropriate temperature for a suitable length of time. Maintain the temperature of the room around 20 degrees Celsius as the temperature affects the growth of the worms at the completion of the chemical exposure in the shaker.

Let the 96 well plates rest for 10 to 15 minutes to allow the adult worms to sediment to the bottom of the plate using a multichannel pipette. Remove 350 microliters of the M nine from each well being careful not to disturb the worms at the bottom of the plate. Wash the worms with one milliliter of M nine and let the plate rest again for 10 to 15 minutes to allow the adult worms to sediment to the bottom.

Remove one milliliter of the M nine from each well being very careful not to disturb the worms at the bottom of the plate. Resus, suspend the worms in the remaining M nine. Collect 100 microliters of the worm M nine mix from the 96 well plate and load it into the wells of a black wall clear Bottom 384 well plate.

Repeat the process using worms from 4 96 well plates until all the wells of the 384 well plate have been loaded. It is important for all the wells to have the same volume in order to increase the efficiency and speed of the autofocus during the image acquisition process. Next, add one microliter of lale to each well and let the worms incubate for approximately 30 minutes.

Lale acts as an acetylcholine receptor agonist and immobilizes the worms transfer the plate to a wide field high content microscope capable of providing automated imaging. Use a high content image acquisition and analysis software according to the manufacturer's guidelines. Select the four x objective to acquire one image per.

Well adjust the GFP imaging parameters to 45 milliseconds of exposure and image resolution to 2160 by 2160. Begin image acquisition exposed worms are directly imaged on 384 well plates. The number of worms containing at least one GFP positive embryo is counted for each well and normalized by the total number of worms in that well as shown here.

Exposure to chemical agents such as the microtubule poison no-code AOL leads to the induction of a high proportion of GFP expressing embryos compared to the DMSO control. The GFP positive embryos are significantly brighter than the weak background fluorescence observed in other embryos, as well as the autofluorescence observed in the gut of the animals. The red arrow indicates GFP positive embryos clearly visible within the uterus of a no-code AOL treated worm.

Following this procedure, other methods can be performed such as DNA staining of the worms in order to get a more detailed look at the effect of the chemicals on the germline. This technique allows researchers in the field of toxicology to quickly assess the reproductive toxicity and germline toxicity of chemicals.