鶏の聴覚脳幹における卵の電気穿孔

The overall goal of this procedure is to spatially and temporally control gene expression in the chicken auditory brainstem. The utilization of in ovo electroporation provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena. This procedure can help you answer key questions in the field of auditory neural development, such as how does the control of gene expression promote either a gain or loss of phenotype that are required for normal auditory brainstem development.

Demonstrating the procedure will be Doctor Ting Lu, a postdoctoral fellow from my laboratory. To begin, prepare plasmid solutions by adding one microliter of Fast Green solution for every seven microliters of DNA mixture. Next, mix DNA solutions at a one-to-one ratio for simultaneous electroporation of multiple plasmids.

Once the plasmid solution is prepared, fabricate pipettes with a micropipette puller. Then use a 28-gauge syringe needle to fill the pipette with one to two microliters of the plasmid solution. Using forceps, break the pipette tip to 10 to 20 microns and determine the size of the broken tip under a microscope, then connect the pipette to a picospritzer via pipette holder.

Prior to the procedure, store eggs purchased from a local vendor for no longer than five days in a refrigerator at 13 degrees Celsius. To begin, disinfect the egg with 70%ethanol, then to achieve proper embryo positioning, incubate the egg on its side at 38 degrees Celsius, and approximately 50%humidity to ensure optimal viability. After 48 to 52 hours of incubation, when the egg reaches Hamburger-Hamilton stage approximately 12 to 13, disinfect the work area and all instruments with 70%ethanol.

Then remove the egg from the incubator, leave it at room temperature, and wipe it with 70%ethanol a second time. Using a light illuminator, identify the positioning of the embryo, visible as a dark area, and with a pencil, mark it by drawing a circle around its center. Poke a hold in the blunt end of the egg with a 19-gauge needle, then make a second hole near the drawn circle on the pointy end of the egg.

Use the 19-gauge needle to remove a small piece of the outer eggshell, and with forceps, remove a small piece of the inner eggshell membrane. Next, introduce a 19-gauge needle attached to a three milliliter syringe through the hole at the blunt end of the egg, and withdraw 1.5 to 2.5 milliliters of albumin. Cover the hole at the blunt end of the egg with a one centimeter by one centimeter piece of tape, and then cover the drawn circle and the second hole with a 2.5 centimeter by four centimeter piece of tape.

Using curved scissors with a cutting edge of 2.5 centimeters, and keeping them parallel to the eggshell, excise a window within the marked circle. To inject the plasmid, turn on the picospritzer and adjust the instrument's setting to apply pressure of 18 PSI for five microseconds, then turn on an air tank, and the lamp for the dissection microscope. Next, prepare 30 milliliters of a stock solution of the commercially purchased India Ink in sterile PBS.

Aspirate the India Ink solution into a one milliliter syringe, then attach a 27-gauge needle and remove any bubbles that appear in the syringe. To visualize the embryo, introduce the needle just under the yolk membrane, approximately two to three millimeters from the embryo, and inject approximately 0.2 milliliters of India Ink. Then transfer the windowed egg to an egg holder, positioned under the dissection microscope, and switch to the highest magnification to achieve the best view of the plasmid injection site.

Remove the membrane covering the injection area with forceps. Find the otocysts, and use them as landmarks to identify rhombomeres five and six, located nearby, from which nucleus magnocellularis and nucleus laminaris arise. Using a micromanipulator, position the pipette containing DNA in the neural tube that overlies the R5-R6 region in-between the otocysts.

Eject DNA into the neural tube with the picospritzer. To begin electroporation, turn on the voltage stimulator, then attach a 0.22 micron syringe filter to a three milliliter syringe pre-filled with PBS, and add one to two drops of the filtered PBS onto the embryo. Subsequently, move a bipolar electrode toward the embryo using the micromanipulator, and position the negative electrode above the injection site, while maintaining the positive electrode lateral to the R5-R6.

With the voltage stimulator, apply 20 pulses of 50 volts for one millisecond at one second intervals. You will see bubbles near the tip of the electrode. After electroporation, add one drop of PBS onto the exposed area, gently remove the electrode and clean it with a laboratory tissue and 70%ethanol, then cover the window over the embryo with tape.

Finally, note the injected plasmid type, and the hatch date on the eggshell, and place the egg in the incubator with window-side facing up. Incubate the eggs at 38 degrees Celsius with 50%humidity, until desired developmental stage is reached. 24 hours after the electroporation, remove the egg from the incubator, open the window, and using a stereo microscope, confirm the correct location of the targeted plasmid injection.

If tet-on vectors were used, pipette 50 microliters of Doxycycline into the chorioallantoic membrane, close the window, and put the egg back into the incubator. Presented here are photographs of a brainstem slice prepared from an embryonic day-12 chicken, taken under differential interference contrast illumination and fluorescent illumination. The nucleus magnocellularis and nucleus laminaris can be recognized within the brainstem slice.

Under fluorescent illumination, both structures reveal the expression of yellow fluorescent protein, confirming efficient spatial expression of the electroporated genes. The expression of the electroporated gene can be also observed in a brainstem slice comprising the nucleus magnocellularis obtained from an embryonic day-18 chicken. When magnified 80 times, the classic adendritic cell bodies are visible under differential interference contrast illumination.

Adendritic cell bodies reveal yellow fluorescent protein expression, further demonstrating that in ovo electroporation can induce gene expression in specific auditory brainstem regions for studies on auditory neuron development. Once mastered, and if it is performed properly, this technique can be done in two to three hours for approximately 12 viable eggs. Following this procedure, other methods like biochemical, pharmacological, and in vitro-in vivo functional assays can be performed in order to answer additional questions about how gene expression phenotypes regulate normal auditory brainstem development.

After watching this video, you should have a good understanding of how to spatially and temporally control gene expression in the chicken auditory brainstem. In ovo electroporation permits genetic control of anatomical and physiological specializations, and well-defined time periods in auditory brainstem development.