The need for enhancers is acquired upon formation of a diploid nucleus during early mouse development.

The activity of the polyoma virus (PyV) origin of DNA replication was used as a sensitive assay for enhancer function in one- and two-cell mouse embryos by injecting embryos with plasmid DNA containing different PyV ori configurations, allowing them to continue development in vitro, and then measuring plasmid DNA replication. Replication always required the PyV origin 'core' sequence in cis and PyV large tumor antigen (T-Ag) in trans. In developing two-cell embryos, DNA replication also required an enhancer in cis. Two copies of part of PyV enhancer 3 (beta element) was sevenfold better than one copy, and enhancer 3 was better than enhancer 1 + 2 (alpha element). Competition between ori configurations suggested that enhancers bound specific proteins required for replication and transcription. In contrast, DNA injected into one-cell embryos did not need an enhancer for replication, and no competition for replication factors was observed between different ori configurations. In fact, ori core replicated about ninefold better in one-cell embryos than the complete origin did in developing two-cell embryos. Therefore, core contains all the cis-acting information necessary to initiate DNA replication. Because one-cell embryos that replicated injected DNA retained their pronuclei and remained one-cell embryos, enhancers are not needed in mammalian development until a diploid nucleus is formed.