A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

The overall goal of this procedure is to modify pitter Miata embryos with perturbing reagents by introducing them via microinjection. This is accomplished by first collecting and maturing gametes from adult animals. The second step of the procedure is to fertilize the mature oocytes.

The third step is to deje the zygotes and place them in rows on an injection dish. The final step is to inject the zygotes with the desired perturbing reagents ultimately introduced foreign reagents such as GFP Reporter can be viewed through fluorescent imaging of the injected embryos. A visual demonstration of this technique is really important because it's difficult to learn how to micro inject, and particularly how to orientate the needle in a three-dimensional space, and how to introduce the correct bolus size into the egg.

So demonstrating this technique today will be a graduate student from my lab, Elise tla. Begin with a collection of animals. Sexing is performed by excision of gametes.

As P Miata, sea stars are not sexually dimorphic. To remove the gonads use a blade to cut a small opening, not more than one centimeter large along the side of an arm. Blunt ended forceps can be used to pull back edges.

Avoid removing the loose, gray, brown gut tissue. Ovaries are orange to light.Brown. Collect them in a small glass dish with a few milliliters of water.

Teasing this soft tissue apart will release the cytes within. Check the quality of cytes to determine if they will mature properly. Then filter the oocytes To remove ovary tissue and small immature oocytes, add five milliliters of 200 micromolar, one methyl lain to 100 milliliters of oocytes in seawater, and allow the oocytes to mature in the solution for 45 to 90 minutes at 15 degrees Celsius.

SA quality is critical for determining whether a culture will mature, fertilize, and develop properly. Only use batches of eggs where most of them are large, brown, clear and yellow, or light brown and colored. Discard batches of embryos where most of em are small.

Brown or greeny Testes are white or beige, and will cloud the water when they are disrupted. Collect the testes in a 1.5 milliliter tube with as little seawater as possible and store them on ice. Begin by mincing up a pea-sized cluster of testis tissue in a milliliter of seawater.

Transfer two or three drops of the resulting milky seawater to 100 milliliters of mature oocytes in a culture dish. This ratio should prevent poly sperm. Do not transfer any of the tissue pieces.

Swirl the mix to GAMTs. After a few minutes, check the GAMTs under a dissection microscope fertilized eggs. Have a fertilization envelope, which looks like a clear bubble around the egg.

Once fertilization is confirmed, change the water by first collecting the zygotes in a 100 micron filter cup, and then washing them off the filter to a clean dish with fresh sea water. Now transfer the zygotes to 15 degrees Celsius for 15 to 30 minutes. In preparation, acidify the seawater to a pH of about four by adding one normal hydrochloric acid dropwise.

Monitor the pH and completely mix in each drop before adding. The next. Also required is a mouth pipette with the opening.

Just a little larger than the diameter of one zygote with a 45 degree angle dent. Now proceed with de jellying the zygotes, so they will stick nicely to the plastic dish. The jelly code is more extensive than that of a sea urchin, but it can be removed with a acidic sea water.

First, filter the zygotes from their water with a 100 micron mesh. Then transfer them to a 200 milliliter beaker with minimal sea water. Then add 150 milliliters of the acidified seawater to the beaker, and let them sit for three minutes.

In the fourth and fifth minute, strip off the jelly coating by pouring the zygotes through a 200 micron mesh with the acidified seawater. Do this five to 10 times. After five minutes have passed.

Collect the deed zygotes on a 100 micron mesh. Then rinse them into a small glass dish with normal seawater. Once in the dish, immediately rode the zygotes before they produce more jelly.

Use aspiration from a mouth pipette to handle the zygotes. Move them from the glass dish to a prepared plastic injection plate. The zygotes are slightly buoyant.

Position the pipettes so that the zygotes are gently pressed onto the surface as they are ejected. An angled end helps in this process. If the zygotes do not stick to the plastic dish, transfer them back to acidic seawater for a few minutes and try again.

Unstuck zygotes will not be injectable ro as many zygotes as desired. Their penetrability does not change over time. Prepare all the injection solutions no matter the makeup to be 200 millimolar potassium chloride.

Morpho oligonucleotide should be at 400 to 800 micromolar and DNA at approximately 2.5 nanograms per microliter heti injection solution with morph ano oligonucleotides to 65 degrees Celsius for five minutes just before their use add 0.1%Rod domine DExT strand tracer to the injection solution for later visualization. An injection needle can be pulled from a one millimeter outer diameter threequarter millimeter inner diameter glass capillary loaded with one to two microliters of injection solution via a micro loader pipette. Then attach it to the manipulator.

Control the ejection with a pico spritzer set to 100 millisecond pulses at 40 pounds per square inch. Use a foot pedal to trigger the ejection angle the pipette at about 60 degrees to the dish surface. Break the tip open by running it gently into the protruding plastic at a scored line on the dish.

Then line it up to an egg. The needle should easily penetrate the zygote. Eject the bolus based on the first injection.

Adjust the ejection duration to deliver a bolus to be about 20 to 30%of the average zygotes diameter. Keep adjusting the duration until the desired bolus is arrived at. Then leave the timing set.

If the needle gets clogged, break the needle again. Then return to a 100 millisecond pulse and readjust the bolus. If an injection duration greater than 200 milliseconds is needed to get the proper bolus, break the needle again.

And if an injection duration needs to be 20 milliseconds or less, replace the needle. If a zygote gets stuck on the needle, remove the needle from the water to remove it. Try to complete the injections before the first cleavage.

Otherwise, only part of the embryo will be affected by the perturbing reagents. Once the cleavage starts, single blasphemers can be injected, which may be ideal for some lines of experimentation using the described technique. A DNA reporter construct expressing GFP was injected into embryonic sea stars.

The injected embryos showed clonal patches of construct expression as expected summary reagent were toxic to the embryos when over injected. Here, an over injected embryo stalled at the one cell stage. Other times the injected material resulted in abnormal division such as an asymmetric early division.

Toxicity could also manifest at aberrant development. After watching this video, you should have a really good understanding of how to culture and micro inject viter mini outer zygotes. You'll create morph an embryos to perform a variety of downstream experiments.