To begin, incubate fertilized chicken eggs in a humidified incubator. Next, prepare two times CMF saline with 0.25%EDTA buffer. Then peel the skin off the chicken embryos in HBSS solution, and incubate them in two times CMF-EDTA solution on ice for 15 to 20 minutes.
Use a pair of watchmakers forceps to carefully separate the epithelium and mesenchyme. Then transfer the isolated epithelium and mesenchyme to a single clean dish containing HBSS. For recombination, place the mesenchyme on a Petri dish containing HBSS, then place the epithelium on it, aligning it along the mesenchyme's anterior/posterior axis.
Alternatively, rotate the epithelium by 90 degrees from the mesenchymal anterior posterior axis. Next, transfer the recombined skins to the culture inserts in six well culture dishes. Remove any excess HBSS surrounding the recombined skin.
After adding DMEM containing 10%FBS to the exterior well of the insert, pipette a thin layer of DMEM into the insert chamber, ensuring the explant remains semi hydrated. Finally, place the skin recombinants in an incubator at 37 degrees in an environment of 5%carbon dioxide, and 95%air. Monitor the phenotypic changes in the initial skin development phases using time-lapse photography, with a mounted dissecting microscope.
New placodes appeared shortly after recombination, and developed into short feather buds two days post-recombination. Long feather buds developed in four days. When the epithelium was rotated by 90 degrees, the orientation of the new buds was determined by the epithelium.
