The overall goal of the following experiments is to perform and manipulate regenerative assays in Parian flatworms using pharmacological and or genetic knockdown methods. The first step is to master the basic amputation and regeneration assay. Next is shown how drugs can be used to subvert regenerative polarity, for example, to yield two-headed animals.
Finally, a description is given of how to perform in vivo RNAi by feeding. In order to examine the effects of loss of function of specific genes. The results show that treatment with the drug quintal subverts regeneration to produce two-headed worms and genetic knockdown of a betaine and isoform produces a grossly similar phenotype.
Hi, my name is John Chan, a graduate student in the marching laboratory, and I'll be demonstrating these procedures. Visual demonstration of these methods is important cause although in itself no individual step is difficult. Overall demonstration of these techniques will assist researchers new to this model system.
In preparation for the assay, stop feeding a cohort of approximately 30 parian for at least five days after starvation. The worm should be between eight and 10 millimeters long on the day of the assay. Rinse a pre-pro leveled ice dish with water and cover the flat iced surface with plastic wrap using forceps.
Place one filter paper on the plastic wrap. Moisten the filter paper with a few drops of spring water using a transfer pipette place no more than 20 plin onto the filter. If necessary, the worms can be repositioned on the filter by re pipetting with more spring water.
Once the worms are in position, remove any excess fluid using a scalpel, amputate the head by making a single cut approximately halfway between the anterior apex of the animal and the anterior end of the pharynx. Next, amputate the tail region by making a single cut approximately halfway between the tail end of the animal and the posterior end of the pharynx. Remove excess mucus on the scalpel using a paper towel moistened with 70%ethanol after cutting, and then wipe excess ethanol from the scalpel before cutting again.
Pick up the filter with forceps and transfer it into a Petri dish containing spring water. Wait approximately three minutes for wound closure, which is observable by a pinching and darkening of the wound. Rinse trunk fragments from the filter paper into a Petri dish containing the desired medium for the regeneration assay and transferred to a thermostat incubator set to 24 degrees Celsius.
Score the regenerative phenotype after about one week. When regenerated structures are identifiable, prepare a 200 millimolar quintal or PZ Q stock by solubilizing the drug in DMSO to use fresh on the same day. Next, make 50 milliliters of drug containing solution by adding 22.5 microliters of the PZ Q stock to buffered majic salts vortex for about one minute.
To ensure dispersion place worms on filter paper moistened with spring water and amputate both the head and the tail region as described in the trunk fragmentation regeneration assay After amputation and wound closure, transfer the trunk fragments into a Petri dish of spring water. Exchange the medium to 90 micromolar PZ Q solution and transfer the Petri dish to the 24 degrees Celsius incubator. After 24 to 48 hours, exchange the PZ Q containing media for spring water, washing the worm several times, return the worms to the incubator and after one week, score the bipolarity or two headedness phenotype of the worms prior to the assay.
Prepare chicken liver homogenate, discard the fatty yellow colored sections from the food stock, and then puree the remaining liver filter puree through a mess. Strainer and aqua spend suspension for storage. Next, prepare bovine red blood cells or RBCs by Ali, quoting the supply into one milliliter samples stored at negative 20 degrees Celsius.
Use worms that have been starved for five days and group them into three cohorts. The gene of interest positive control a gene with known RNAi phenotype and negative control. A gene with no RNAi phenotype.
For each cohort, use about 250 worms that are eight to 10 millimeters long. Thaw a stock of e coli for each cohort that expresses double stranded RNA, targeting the genes of interest along with a tube of chicken, liver, homogenate, and a tube of RBCs. Pellet the bacteria by centrifusion at 13, 000 Gs.For one minute, remove the supernat and resuspend the pellet in 700 microliters of two XYT media.
Re centrifuge, discard the supernatant and then place the pellet on ice. Create a large bore P 200 pipette tip by cutting off the tip thoroughly resuspend the bacterial pellet in 150 microliters of chicken liver homogenate plus 50 microliters of RBCs to create an RNAi feeding mix. Remove air bubbles by a short centrifugation pulse for feeding.
Remove a majority of the water from the plastic tub containing worms, leaving about one inch depth. Swirl the tub to concentrate worms at the center of this container. And then pipette RNAI feeding mix in a circle, corralling the worms.
Use a transfer pipette to carefully coax any escapees back onto the RNAi feeding mix without perturbing the other diners after feeding for approximately one hour, identify parian that fed well and remove any worms that do not appear well fed. Well fed worms. Show a deep red coloration from ingested RBCs.
Carefully replace the feeding solution with fresh spring water, being careful to minimize disturbance in order to prevent ingestion of food. To do this gently localize the worms to one side of the tub using a transfer pipe. Pour out the turd water and carefully replace with fresh water poured down the opposite wall of the tub res submerge parian quickly as prolonged exposure to air.
Also results in food ingestion loosely. Seal the container lid and return the worms to the 24 degrees Celsius incubator. Repeat this RNAi feeding protocol over multiple cycles interspersed with regenerative cycles to ultimately screen for RNAi phenotypes.
A standard feeding and regeneration schedule that is effective for many genes is shown here. F represents the feeding protocol and X represents the regeneration assay. In total, the protocol takes about one month and it can be modified for different genes by including fewer, represented by parentheses F or greater feedings, as the optimal protocol will depend on factors such as mRNA stability, tissue localization, protein, P dur, or the development of a phenotype that precludes multiple feeding cycles after regeneration.
Here are results typical of the trunk regeneration assay. On the left, a parian is shown above a schematic indicating the amputation cuts. On the right is a time course of trunk fragment regeneration that takes place over seven days with eye spots appearing by day three.
Here, the pharmacological subversion of trunk fragment regeneration by PZ Q.Treatment has yielded two-headed worms using RNAi against the gene DJ 61. In an FX FX feeding protocol results in an ilis phenotype, the loss of a light aversion response seen after an FFFX feeding protocol results in immobile DJ PC two RNAi worms stained red compared to mobile control. Worm stained green RNAi knocked down of DJ beta catine in one.
Using an FXFX feeding protocol results in the regeneration of a head from the posterior wound. Following these procedures, researchers will be able to target genes of interest to their research using either knockdown methods or selective pharmacological agents in order to explore their role in parian biology and tissue regeneration.
