Generation of Transgenic Hydra by Embryo Microinjection

The overall goal of this procedure is to micro inject plasma DNA into hydra embryos to produce transgenic lines. This is accomplished by first preparing a plasma DNA solution and loading it into an injection needle. Next, the plasma DNA solution is injected into hydra embryos between the one and eight cell stages.

Then the hydra embryos are allowed to develop and hatch and juveniles with transgenic tissue are selected. The final step is to produce a uniform transgenic line by asexual budding of the original transgenic animal. Ultimately, fluorescence microscopy is used to visualize transgenic expression in hydra.

This method was designed for researchers who wanna make transgenic hydro lines at low cost, thus allowing for a wider application of this useful technology, both for current hydro researchers, but also those that are just wanna start using this model. Transgenic hydra are used to study tissue dynamics, protein localization gene function by knockdown and overexpression, and also promoter function. After preparing plasma DNA according to the text protocol, prepare an injection dish by first placing a 75 by 50 glass microscope.

Slide into a 100 by 15 millimeter Petri dish at an angle dissolve 2%agros in 50 milliliters of hydra media. Let it cool. Then pour it into the Petri dish After the agros has solidified, remove the glass slide and with the razor blade, cut away any excess agros to form a wall.

Then immediately pour hydra medium into the dish and either use it immediately or store it at four degrees Celsius until needed. To prepare the injection solution, combine three microliters of 2.5 milligrams per milliliter plasma DNA solution with two microliters of 10%phenol red solution. Vortex the solution briefly and then spin it for 10 minutes at maximum speed in a micro centrifuge to remove any particles that might clog the needle under a dissecting microscope.

Use forceps to clip the needle a few millimeters from the tip to create a small opening. Orient the Petri dish containing the needle so that the needle is in a vertical position with the tip down. Then pipette approximately 0.5 microliters of injection solution into the back end of the needle.

To prepare embryos for injection, begin by scanning a hydra, a EP strain culture for polyps producing eggs. Collect these polyps and transfer them into 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 dermal cells, they are ready for injection place polyps that have eggs ready for injection in a dish with several hydra that have testes for at least an hour before injection to allow for fertilization if females are found in the a EP colony with fully formed eggs or one to eight cell stage embryos. Also collect these for injection before injection. Use a scalpel with a number 15 blade to remove most of the parental tissue above and below embryos that have begun to cleave, leaving them with only a small piece of the parent's body column attached.

Save the dissected tissue in Hydra medium as it will regenerate and ensures that females remain in the hydra colony. Use a pasture pipette to transfer the cleaving embryos to an injection dish, arranging them parallel to the wall of the agros trough. To prepare the micro injector mounted on a magnetic stand that sits on an iron plate.

Mount the injection holder, which is part of the micro injector that holds the needle on a joystick microm manipulator. Using a magnetic stand mount the joystick manipulator onto the iron plate. Then place the entire setup on the right side of a dissecting microscope and position the injection holder so that it is visible in the observation field.

Place the injection dish with embryos under the dissecting microscope oriented such that the vertical wall of the agros 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. Next, 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. Center an embryo in the field of view, and move the needle so that it is touching the embryo. Then use the microm manipulator to pierce the embryo with the needle and allow the injection solution to flow into the embryo for one or two seconds before quickly removing the needle.

If the embryo has more than one cell, inject each cell individually. Continue until all the embryos have been injected. Once all the embryos have been injected, transfer them into a dish with a few Hydra, a EP polyps that have testes and incubate them at 18 degrees Celsius while they continue to undergo embryogenesis.

When the embryos reach the cuticle stage, move each embryo into a well of a 24 well plate filled with hydra medium, and incubate for two weeks in the dark at 18 degrees Celsius. Then under a dissecting microscope, check each embryo for detachment from the small piece of parental tissue and the regeneration of that tissue. If present, remove it immediately so it is not mistaken for a new hatchling.

After checking each embryo, move the dish under an aquarium light at room temperature. Check the plates each day and collect the hatchlings. New hatchlings will be white in color because they have not eaten yet.

Observe them under a fluorescent microscope for transgene expression. Use art napoli to feed transgenic hatchlings every two to three days. When hatchlings that are transgenic in either the electrodermal or endodermal epithelial tissue produce buds, they are assessed for transgene expression.

Shown here is a hatchling with chimeric expression of a Ds red, two transgene under the control of an actin promoter and a patch of ectodermal epithelial cells. Continuous budding by the animal can eventually produce a line with uniform expression of the transgene in either the electrodermal or endodermal epithelial lineage. As seen here, the original hatchling is now producing two new buds.

The bud labeled with an asterisk, has no transgenic tissue and was used as the founding animal for a control line that is genetically identical to the transgenic line except for the presence of the transgene. This animal is the first bud with transgenic tissue produced from the original hatchling. Through these experiments using transgenes that have no impact on biological function, uniform epithelial cell lines were established from approximately 30%of hydra that hatch with epithelial transgenic tissue.

A uniform electrodermal line as seen here is established 33 to 50%less often than an endodermal line. So after watching this video, you should have a good understanding of how to make transgenic hydro lines, which will add to the repertoire of functional studies you can do with this model.