Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

Using microinjection to deliver DNA constructs into Strongyloides allows us to generate knockouts and transgenics, which can be used to study how these worms develop, navigate their environment, and parasitize hosts. So far, this is the only successful technique for generating knockouts and transgenics in these parasites. One day before microinjection, add 180 microliters of the agarose solution onto a cover slip using a glass Pasteur pipette, then immediately drop a second cover slip on top to flatten the agarose into a thin pad.

After 5 to 10 seconds, remove the top cover slip by sliding the two apart and determine which slide the agar pad is on, and lay it face up. Then, select a tiny piece of glass shard from a broken cover slip, and using forceps, gently press it into the agar near the top edge of the pad. On the day of the microinjection, line the Baermann holder, which is a sieve made from two plastic rings with two layers of nylon tuile netting secured between them with three overlapping pieces of lab tissue.

Then add the fecal-charcoal mixture to the Baermann holder. Next place the Baermann holder with the fecal-charcoal mixture in the funnel, fold the pieces of tissues around the fecal-charcoal mix, and add enough water to submerge most of the fecal charcoal. Then, top the funnel with a 15-centimeter plastic Petri dish lid to contain the odor and label the funnel as needed.

After one to two hours, hold a 50-milliliter centrifuge tube under the rubber tubing at the bottom of the funnel and carefully open the clamps at the bottom to dispense 30 to 40 milliliters of water containing worms into the 50-milliliter tube.Then. transfer 15 milliliters of the Baermann water containing the worms to a 15-milliliter centrifuge tube. Spin the tube, remove up to 13 milliliters of the supernatant, and discard it into a liquid waste container with iodine to kill any worms.

After collecting all the worms, inspect the pellet of worms at the bottom of the tube. If no worms are visible, wait for one to two hours then collect more worms from the Baermann apparatus. Transfer the worms in as little water as possible to a six-centimeter 2%nematode growth medium plate with a lawn of E.coli HB101 and use this plate as the source plate for the microinjection.

Under the dissecting microscope, cover the shard of glass on the microinjection pad cover slip with halocarbon oil using a standard platinum worm pick. Then, place the microinjection pad cover slip on the microinjection scope and locate the shard of glass covered in oil. Align the glass shard such that an edge is perpendicular to the direction of the needle to serve as the surface used to break the needle.

Next, using a dissecting microscope, ensure that the needle has no bubbles or debris in the tapered shaft, then secure the needle 1 to 1.5 centimeters into the pressurized holder. Position the tip of the needle in the center of the microscope field-of-view by eye and under low-magnification, position the tip of the needle in the field-of-view perpendicular to the side of the glass shard. Then, switch to higher magnification and align the tip of the needle with the edge of the glass, near, but not touching it.

Next, to break the tip of the needle and allow liquid flow, gently tap the needle on the side of the piece of glass while applying continuous pressure from the gas. Once the liquid begins to flow, check the shape of the tip and ensure that it is sharp with easily flowing liquid. When the liquid is flowing well from the needle, move the microinjection slide to the dissecting scope and add one to two microliters of halocarbon oil on the agar pad for placement of the worms.

Transfer 20 to 30 young adult Strongyloides to a 2%nematode growth medium plate without bacteria for at least five minutes to remove excess surface bacteria and select single worms for microinjection. Add more worms to the plate as needed while injecting. Using a small amount of halocarbon oil on a worm pick, select a Strongyloides young adult female with one to four eggs in her gonad from the plate without bacteria and transfer the worm into a tiny drop of oil on the agar pad.

Using the worm pick, gently position the worm so it is not coiled and the gonad is visible and easy to access. Then, position the worm in the microinjection microscope field-of-view, ensuring that the gonad is on the same side as the needle and positioned so that the needle will contact the gonad at a slight angle. Next, bring the tip of the needle to the side of the worm in the same focal plane.

Then, aiming for the gonad arm near the middle of the worm, gently insert the needle into the gonad using a microinjector. Immediately apply pressure to the needle to gently fill the entire gonad arm with the DNA solution. After enough fluid has been injected, remove the needle and observe that the wound closes.

Repeat the procedure with the other arm of the gonad, if it is visible. Once the injection is completed, quickly verify that the needle is not clogged by applying pressure with the tip of the needle on the agar pad, then transfer the slide with the injected worm to the dissecting microscope. To recover the injected worm, first place a few drops of worm-buffered saline on the worm to float it off the agar pad.

Then collect a small amount of bacteria on a worm pick and touch the worm with the adherent bacteria to remove it from the liquid. Gently transfer the worm to the recovery plate. For behavioral experiments, transfer 20 to 30 larva to a 2%nematode growth medium plate with a thick HB101 bacteria lawn, and under a fluorescence dissecting microscope, identify the larvae expressing the transgene of interest.

Then, using a worm pick, select the transgenic larvae and place them in a small watch glass with worm-buffered saline. For microscopy, using a razor blade, score a grid onto the plastic bottom of a 10-centimeter chemotaxis plate to easily track the location of the worms on the plate. Next, add three microliters of larvae in worm-buffered saline into a square on the grid, filling as many squares as needed.

Then add 15-to 20-microliter drops of 1%nicotine in water to the worm drops. After four minutes, when the worms are paralyzed, screen them using a fluorescence dissecting microscope. Transgenic Strongyloides stercolaris larvae with an incomplete and complete act-2 mRFPmars expression pattern are show here.

The patchy expression in the body wall muscle is more common when the transgene is not integrated into the genome. In contrast, consistent expression throughout the body wall muscle often indicates that the transgene has integrated into the genome. The key step is positioning the needle in the same plane of focus as the gonad to ensure the DNA is injected into the gonad and will be incorporated into the developing eggs.

The development of this technique made it possible to study gene function in parasitic nematodes, which is providing new insights into mechanisms of parasitism and host-parasite interactions.