A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs

Stage X chick blastoderms following oviposition were accessed via a small window in the egg. Windowing, however, substantially reduces the hatchability of eggs containing early embryos. For example, only 32 of 389 (8.2%) eggs hatched after standard windowing with or without irradiation or injection. Ex ovo culture systems can overcome this problem but are labor intensive. A modification of a standard windowing technique has yielded an average hatch rate of 32% for 892 windowed eggs independent of incubator type, gamma-irradiation, or injection of the embryo. This was a fourfold increase over a standard windowing method. Similar hatch rates were observed using fertile eggs from five chicken lines [Barred Plymouth Rock (BR), Athens-Canadian (AC), Line 0, SPAFAS, and commercial White Leghorns (WL)]. The modification involves covering the egg shell membrane with PBS after grinding away the shell and before piercing the membrane. The window is then sealed by overlaying with fresh shell membrane and cementing it in place once it has dried. The method has been used successfully for the production of somatic and germline chimeras because donor BR blastodermal cells injected into Stage X, gamma-irradiated recipient embryos from WL or AC yielded a hatch of 33.7%, of which 42.3% were feather chimeras. Two of 11 cockerels tested were germline mosaics bearing at least 1% BR sperm. The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development.