Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

The overall goal of this procedure is to perform large scale RNA interference screens in C elegance using a bacterial DS RNA feeding library to knock down gene expression. This is accomplished by first culturing bacterial clones from a DS RNA library. Under growth conditions that prevent plasmid loss begin by duplicating each primary library plate and then using the 96 well duplicate library plate to generate temporary stalks of bacteria on agar omni plates.

Bacteria from these omni plates are grown overnight in liquid culture and subsequently transferred to the surface of worm feeding plates. Each bacterial clone from the library carries a unique D-S-R-N-A encoding plasmid that targets one C elgan gene for knockdown. The second step is to place synchronized worms onto the feeding plates.

Next, the worms are grown for several days in a humidified chamber at 20 degrees Celsius. As the worms feed on the bacteria, the induced DS RNAs are released into the gut of the animal where it then spreads to surrounding tissues to cause systemic knockdown effects. The final step is to examine the fed animals for phenotypic effects caused by gene specific knockdown.

These phenotypic assays are used to identify genes important for the biological process under study. The main advantage of this technique over existing methods like microinjection of doublet stranded RNAs into the worm gonad is that it's high throughput. Using feeding libraries, we can examine the biological function of nearly all sea elgin's genes in just a matter of weeks.

Many labs have struggled with this knockdown approach because most genes and C elegance are haplo sufficient. And so RNA knockdown must decrease gene expression by greater than 50%To produce loss of function phenotypes. To ensure robust gene knockdown, it is critical that all of the bacteria fed to the worms express the double stranded RNA plasmid.

To begin the procedure for determining plasmid loss, remove a small sample of bacteria as described at each relevant step and add it to 450 microliters of sterile water. Use 100 microliters of this diluted sample to create further serial dilutions using sterile water. Mix the solutions thoroughly between dilutions.

Spread 100 microliters of each dilution onto LB and LB AMP plates and incubate overnight at 37 degrees Celsius on the following day, identify the LB plate that contains between 100 and 500 bacterial colonies. Count the number of colonies on this plate and on the corresponding lb amp plate containing the same dilution of bacteria. Determine plasmid loss using this equation.

The percentage of bacteria that do not contain the plasmid is calculated by subtracting the number of colonies on the LB amp plate from the number of colonies on the LB plate, dividing by the number of colonies on the LB plate and multiplying by 100 Plasmid loss is prevented by growing bacteria in high concentrations of carbonic sellin For effective knockdown. Check for plasmid loss before feeding animals and continue only if plasmid loss is less than 15%Begin this procedure by removing duplicate library plates from the negative 80 degree Celsius freezer. Take off the foil seal while the cultures are still frozen.

Replace the plates plastic cover and set the duplicate library plates on a level surface to thaw at room temperature for no longer than 30 minutes. Sterilize the buccal 96 pin replicator. Place it into an ethanol bath and then burn the ethanol.

Coating the pins off using a bun and burner. Allow the flame to extinguish and then repeat. The pins of a clean replicator should be rinsed with distilled water and then sterilized before each use.

Place the sterilized replicator into the wells of the thawed duplicate plate and use it to gently stir the cultures. Small volumes of the culture will adhere to the pins of the replicator. Carefully remove the replicator from the wells of the duplicate plate and place it gently pins down on the surface of the omni plate.

Be careful not to pierce the agar surface. Leave the replicator on the surface of the omni plate for three to four seconds so that bacteria from the pins of the replicator are transferred onto the agar surface. To prevent plasmid loss on solid media, the ate contains two milligrams per milliliter.

Carbonic sellin. Remove the replicator and allow the small volume of bacterial culture to absorb into the agar. After both plates have been duplicated.

Incubate the omni plates inverted for 15 to 18 hours at 37 degrees Celsius on the following morning. These omni plates can either be used to inoculate 96 well liquid cultures or stored up to seven days at four degrees Celsius. Bacteria for feeding worms are prepared as liquid cultures using bacteria from the omni plates using a repeat pipetter and a 50 milliliter combi tip, dispense one milliliter of bacterial growth media into each two milliliter.

Deep well of a 96 well plate sterilize the replicator and place it onto the surface of an omni plate containing bacterial colonies, making certain the pins are in contact with each of the 96 bacterial colonies. Carefully move the replicator from the surface of the omni plate into the deep well plate, making certain that the pins do not scrape the sides of the deep wells until immersed in growth media. To dislodge the bacteria into the growth media.

Move the replicator slowly in a square motion following the inside wall of the deep well plate. Be careful not to splash and cross contaminate wells. For each 96 well deep well culture plate two controls should be included.

Bacteria expressing DS RNA as a positive control for the expected knockdown, phenotype and bacteria containing empty DS RNA vector as a negative control. Using a sterile inoculating loop, remove a single well isolated colony from the positive control plate and inoculate an empty well in the deep well called plate. Record the position of the well that has been inoculated.

Repeat for the negative control. Shake the deep well plates in a flat position at 650 RPM on a micro shaker at 37 degrees Celsius overnight. After overnight incubation of cultures, the bacteria from the 96 deep well plates will be transferred to 12.

Well feeding plates transfer can be done four wells at a time. Place pipette tips on every third channel of a 12 channel multi-channel pipette. Remove 150 microliters of bacterial culture from the deep well plate and eject onto the surface of four wells of the 12 well feeding plate.

It is important not to pierce the surface of the agar during transfer as worms will burrow and not feed on the DS RNA expressing bacteria. After each set of four wells is transferred, replace the four pipette tips with four new sterile pipette tips and seed the next four wells of the feeding plate store the seeded plates upright in the dark at room temperature overnight so the bacteria can absorb into the agar. Prior to transferring L one arrested C elgan larvae onto DS RNA feeding plates for a feeding screen.

The concentration of the previously prepared L one arrested larvae must be determined. Mix the erlenmeyer flask containing the arrested L one larvae to distribute the animals. Then remove a 10 microliter aliquot and place dropwise onto an unseated six centimeter NGM plate.

In four to five drops down the center of the plate. Make sure that all media is expelled from the pipette. Using the end of the pipette tip, spread the dots of worm suspension to form a single continuous line of liquid that spans the diameter of the plate.

Using a dissecting microscope count all animals starting at one end of the line of liquid and continuing to the other end before the liquid has absorbed into the plate and the animals disperse. This may require constant refocusing of the dissecting scope to be certain that no animals are missed. Repeat this counting procedure for three separate 10 microliter aliquots to determine the average number of worms per microliter of worm suspension and record this number directly on the erlenmeyer flask.

To begin the feeding screen, use the suspension of L one arrested larvae and sterile water to prepare a solution containing 2000 worms per milliliter. Each 12 well feeding plate will require 120 microliters of this worm suspension mixed thoroughly to ensure an even distribution of animals and transfer the animals to a sterile reservoir for pipetting. Using the multichannel pipette set up with tips in every third position, transfer 10 microliters of the worm solution directly onto the bacterial lawn of the feeding plates.

Take care not to pierce the surface of the agar as worms will burrow into the agar and not feed on the D-S-R-N-A expressing bacteria. After transfer of worm solution to every two rows of feeding plates, remix the worm solution in the reservoir by gently sloshing it about as worms settle quickly examine a few wells early on under the dissecting scope to ensure that each well is getting 20 to 30 worms. Once all feeding plates have worms, store plates upright in a humidified box in a 20 degrees Celsius incubator the next day after the worm suspension has absorbed into the plate, invert the plates and return to the humidor at 20 degrees Celsius.

Animals on these plates can be observed after three days for P zero phenotypes and then again after six days for F1 phenotypes. The protocol described here was used to test four genes for knockdown egg L 30 dumpy 17, PAT 10, and on four egg L 30 and on four are expressed in the nervous system. Pat 10 is expressed in body wall muscle and dumpy 17 is expressed in epidermal cells.

Erie one Lin 15 B mutants were used in the screens because of their enhanced sensitivity to RNAi as a negative control. The worms were fed bacteria containing the empty PL 44 40 plasmid that did not express a DS RNA as expected. RIE one Lin 15 B animals fed bacteria containing in the empty plasmid did not show any morphological or behavioral defects.

This photograph shows freely moving animals as evidenced by their normal body posture. The black arrow indicates the position of a young adult animal. The white arrow indicates the position of a group of laid eggs.

The normal behavior of the worms is shown in this video clip egg L 30 encodes the sea elegance ortho log of the G-protein subunit G alpha Q.When L one stage Erie one Lin 15 B larvae were fed bacteria expressing egg L 30 DS RNA. The larvae grew to become adults that showed clear defects in locomotion and egg laying behavior. After three days, 100%of the animals fed DS RNA against egg L 30 were paralyzed and unable to lay eggs.

This photograph shows that all animals have adopted a rigid appearance typical of paralyzed animals. The complete absence of laid eggs on the plate should also be noted. Dumpy 17 encodes a collagen protein required for cuticle formation in sea.

Elgan and dumpy 17 null mutants are shorter and fatter than wild type animals. In this experiment, no morphological defects were observed in P zero animals fed dumpy 17 D-S-R-N-A. However, 98%of the F1 animals as shown in this figure showed the dumpy phenotype.

Note that adult animals in the field, one of which is marked by the arrow, are shorter and fatter than the adult animal marked by the black arrow. In panel A.The lack of short and fat P zero animals indicates that dumpy 17 gene function is required at early larval stages for proper body morphology. Pat 10 encodes the body wall muscle troponin CA protein containing four EF hand motifs that bind calcium.

It was observed in this study that 100%of Erie one Lin 15 B animals fed Pat 10 DS RNA became paralyzed within three days and laid no eggs leading to a lethal phenotype. Note that although the animals are paralyzed, they can still move their head muscles to feed as indicated by the clearing of bacteria near the head marked by the arrow on four encodes, a homeo domain protein expressed in ventral cord motor neurons and is required for proper synaptic input. Choice on four was chosen as a test gene in this study because it has proven to be resistant to previous DS RNA feeding strategies.

Results from this study showed that 62%of animals fed on four DS RNA expressing bacteria from the library preparation showed defects in locomotion behavior compared to wild type animals that back freely in a sinusoidal motion when prodded on the head, UNC four null mutants do not back, but instead contract their tightly causing a dorsal flexor, which often becomes so extreme that the head and tail of the animal touch placing the body in a coiled position. This table shows the percentage of animals that exhibit null like phenotypes when fed DS RNA against each of the four genes tested 100 animals were examined for each knockdown experiment. These results demonstrate the importance of plasmid retention for the success of DS RNA.

Interference screens robust and highly penetrant loss of function phenotypes can be observed in all tissues. When all of the bacteria fed to worms express DS RNA Once mastered, the entire feeding library can be screened by one person in about two months. To improve throughput, we only test each bacterial clone once in a feeding screen.

Any genes identified in the first pass through the library are then retested. In triplicate, we insist that the desired phenotype is observed in all retests for the gene to be squared as a positive hit. The plasmid is then purified from the bacteria and sequenced to verify the identity of the encoded gene.

Once a gene is identified, we order a L mutant if one's available and test it for the desired phenotype before performing any additional characterization of the gene.