The overall goal of this method is to perform genetic manipulations in the developing chicken auditory organ from embryonic day four, onward. This is significant because of the difficulty of working with the advanced developmental stage of the chicken embryo. The main advantage of this technique is that it specifically targets the developing auditory organ in ovo with relative ease compared to other techniques which require complex in utero surgeries.

This method can help answer key questions of auditory sensory development. Including how the spatial and temporal pattern of sensory progenital cell proliferation and herital differentiation are coordinated. To begin this procedure attach the glass capillary needle using the tubing connected to the glass syringe which is placed onto the right-hand micromanipulator, and place the electrodes onto the left-hand micromanipulator.

Under the microscope, cut the needle tip using a pair of forceps. Next, place 2.5 microliters of plasma DNA tinted with 0.1%fast green into a dish. Fill the needle with plasma DNA using the plunger of the syringe attached to the right-hand micromanipulator.

Place an egg on its side in a holding device. Then, cut a round window on top of the egg using scissors. After that, place the egg in the holding device under the microscope.

Carefully open the two membranes overlaying the embryo using a pair of forceps to expose the right otic vesicle. While stabilizing the head of the embryo with forceps using the left hand, deliver approximately 0.5 microliters of plasma DNA with the right-hand micromanipulator. Be careful not to inject past the otic vesicle lumen because this will damage the otic vesicle.

Microinjecting DNA into the otic vesicle lumen without damaging the otic vesicle is critical and it requires the use of both hands. Immediately after microinjection, use the left-hand micromanipulator to place the two millimeter platinum positive and negative electrode set anterior ventral to the right otic vesicle, and deliver four pulses at 12 volts by operating the electroporador. The precise placement of electrodes onto the anterior ventral presumptive auditory organ area is important and ensures that the developing auditory organ is targeted.

Immediately after electroporation, add 50 microliters of EdU onto the whole embryo in ovo. Next, seal the egg with tape. Return all the manipulated and sealed eggs to the incubator for 18 to 96 hours.

After 18 to 24 hours, carefully remove the tape. Check the embryos for fluorescent GFP or RFP signal within the otic vesicle using a standard fluorescent microscope. If fluorescent signal is present, harvest the embryos for analyses or re-seal them with tape and return them to the incubator for harvesting and analyses at later stages.

RNA in situ hybridization was used to determine whether electroporation successfully targeted the auditory portion of the developing inner ear. Within the developing BP, sensory progenitors were identified by their expression of Sox2 and Lunatic Fringe. These images demonstrate that sensory progenitors throughout the developing BP at the base and apex were successfully targeted and expressed GFP after 68 hours of electroporation.

In addition, GFP was expressed outside the sensory domain in non-sensory epithelial cells and in the auditory ganglion marked by Neurod1 expression. To determine the proliferative behavior of sensory progenitors, EdU, a thymidine analogue, was added at the time of electroporation at E four, and four days later, EdU incorporation in the developing BP was analyzed in tissue sections. Immunolabeling for the hair cell-specific protein, Myosin7a, was used to identify differentiating hair cells in the developing BP.In the apical portion of the BP, Myo7a positive hair cells were frequently EdU positive.

Whereas in the base, only few Myo7a positive hair cells had incorporated EdU, suggesting that at the time of EdU addition at E four, sensory progenitors in the base are already largely postmitotic. Once mastered, this technique can be completed in five to eight minutes per egg. While performing this procedure, it is important to use freshly prepared and sterile reagents to ensure the survival of the embryos.

Following this procedure, the method parameters can be modified to study other phenomena in the developing inner ear, such as the development of vestibular organs and inner ear neurogenesis. This technique paves the way for researchers in the inner ear field to specifically study the relatively late developing chicken auditory organ from embryonic day four, onward. After watching this video, you should have a good understanding of how to appropriately handle the chicken embryo, conducting microinjection and electroporation, and conduct the in ovo cell proliferation assay.