1. Preparation of Plasmid DNA, Needles, and Injection Dishes

1. Prepare the plasmid DNA using a High Speed Maxi or Midi kit and concentrate the DNA to 2.5 mg/ml using ethanol precipitation. The DNA pellet should be dissolved in nuclease-free deionized water. Store the DNA in 10-20 µl aliquots at -20°C. (See Supplementary Table 1 for a list of available vectors)
2. Make an injection dish using agarose to construct a trough for holding embryos in a 100 x 15 mm Petri dish.
   1. Place a 75 x 50 glass microscope slide into a 100 x 15 mm Petri dish at an angle. Pour approximately 50 ml of 2% agarose melted in Hydra medium²⁴ into the Petri dish. Alternatively, pour the agarose into the dish first and float a “Microinjection Fish Mold” on top.
   2. After the agarose has solidified, remove the glass slide or the mold. If a glass slide was used, cut away excess agarose with a razor blade to form a wall. Pour Hydra medium into the dish and use it immediately or store it at 4°C until needed. Note: Injection dishes can be reused if stored at 4°C between uses.
3. Pull needles from borosilicate glass capillaries with a filament on the micropipette puller using the following conditions: heat 525, pull 75, velocity 100, time 50. Store the needles by pressing them into a narrow strip of clay in a Petri dish such that they are held parallel to the bottom of the dish.
4. To prepare the injection solution, combine 3 µl of 2.5 mg/ml plasmid DNA solution with 2 µl of 10% phenol red. Vortex the solution briefly and then spin it for 10 min at maximum speed in a microcentrifuge to remove any particles that might clog the needle.
5. Under a dissecting microscope, clip the needle a few millimeters from the tip with forceps to create a small opening. Note: There is some flexibility in the appropriate size of the opening because the pressure can be adjusted in step 3.3 to accommodate this variation.
6. Place the Petri dish so that it is holding the needle in a vertical position with the tip down. Load the needle with approximately 0.5 µl of injection solution by pipetting the liquid into the back end of the needle. Allow capillary action to pull the solution into the tip of the needle.

2. Preparation of Embryos for Injection

1. On a daily basis scan the Hydra AEP strain culture for polyps producing eggs. Collect these polyps and set them aside in a separate culture dish. Observe these polyps every few hours during the day to monitor the progress of egg formation. When eggs break through the ectoderm and sit on a ring of retracted ectodermal cells they are ready for injection²⁵. Place these polyps in a dish with several Hydra that have testes for at least 1 hr before injection to allow for fertilization. Hydra males will release sperm without any special manipulation.
2. If females are found in the AEP colony with fully-formed eggs, which are approximately 400 μm in diameter²⁵, or 1- to 8-cell stage embryos, also collect these for injection. Immediately inject embryos that have started cleaving. Note: It is not possible to tell the difference between fully-formed eggs and single-celled zygotes, thus these should be incubated with males prior to injection.
3. Before injection, remove most of the parental tissue above and below the embryo using a scalpel with a #15 blade. Leave only the embryo with a small piece of body column attached, to be used to manipulate the embryo with forceps if necessary. Note: The tissue that is dissected away from the embryo will regenerate and should be saved to ensure that females remain in the Hydra colony.
4. Using a Pasteur pipette, move embryos to the injection dish, arranging them parallel to the wall of the agarose trough.

3. Microinjection of Plasmid DNA

1. Mount the microinjector on a magnetic stand that sits on an iron plate. Mount the injection holder, which is the part of the microinjector that holds the needle, on a joystick micromanipulator. Mount the joystick manipulator to the iron plate, with a magnetic stand. Place this entire set-up on the right side of a dissecting microscope and position the injection holder so that it is visible in the observation field.
2. Place the injection dish with embryos under the dissecting microscope, oriented such that the vertical wall of the agarose trough is to the left. Insert the needle into the injection holder and lower the tip of the needle into the Hydra medium in the dish.
3. Slowly turn the knob of the syringe in the clockwise direction until mineral oil fills the top of the needle and a steady stream of injection solution is observed exiting the needle into the Hydra medium. If the stream is too strong, lower the pressure by turning the knob counterclockwise. Practice this step to routinely obtain a suitable pressure, which depends on the size of the needle opening (i.e., how the needle was clipped in step 1.5). Note: If the stream is too strong, the embryo will not survive the injection.
4. While viewing through the dissecting microscope, move the injection dish so that the first embryo is at the center of the field. Move the needle so that it is touching that embryo. Use the micromanipulator to pierce the embryo with the needle. Note: The vertical wall of the agarose trough will keep the embryo from being pushed aside by the needle.
5. Allow the injection solution to flow into the embryo 1 or 2 sec and then remove the needle quickly. If the embryo has more than one cell, inject each cell individually. Use forceps to reorient the embryo to align the next cell with the tip of the needle.
6. After the first embryo is injected, move the dish so that the next embryo is in the injection position and repeat the procedure; continue until all embryos have been injected.

4. Culturing and Hatching of Embryos

1. Move all of the injected embryos into a dish with a few Hydra that have testes (this is to ensure that all embryos are fertilized). Incubate the embryos in an 18 °C incubator while they continue embryogenesis.
2. Once the cuticle stage is reached, move each injected embryo to an individual well of a 24-well plate filled with Hydra medium.
3. Incubate the injected embryos for 2 weeks in the dark at 18 °C.
4. After the 2 week incubation period, check each injected embryo under the dissecting microscope. Note any cases in which the cuticle-stage embryo has detached from the small piece of parental tissue and the parental tissue has regenerated. Remove this regenerated polyp immediately so that it is not mistaken for a new hatchling.
5. Move the dish of injected embryos under an aquarium light at RT. Check the plates each day and collect hatchlings. New hatchlings will be white because the typical pink color of Hydra polyps comes from the Artemia they eat.
6. Observe the hatchlings under a fluorescence microscope to detect transgene expression.