ABSTRACT
We have developed a method for culture of the chick fertilized ovum to hatch. This method enables access to the earliest stages of embryo development, which normally occur within the oviduct of the hen. The procedure has three stages, to allow for the differing requirements of the embryo. The fate of plasmid DNA injected into fertilized ova prior to the first cleavage division was followed by culturing injected embryos for up to 7 days. Southern transfer analysis of total DNA extracted from injected embryos suggested that the introduced DNA replicated during the first 24 h of development and was subsequently gradually lost. The results of injecting the construct pHFBGCM, which contains the lacZ gene under control of the CMV immediate-early promoter, have been followed by staining for β-galactosidase activity. Expression of the reporter gene was first seen after 12 h of development. After 24 h, at the onset of blastulation, more than 90% of embryos contained stained cells within the area pellucida. The number of cultures with stained cells within the embryo, rather than in the extraembryonic blastoderm, decreased substantially after the primitive streak stage. After 7 days in culture, only 7% of surviving embryos had stained cells within embryonic tissues. These results suggest that, if plasmid DNA is integrated into the host chromosomes, integration is a relatively rare event. Possible future directions for development of the system described are discussed.
