Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis.

We have analyzed the fate of circular and linear DNA molecules following microinjection into the cytoplasm of fertilized eggs of Xenopus laevis. Recombinant plasmids containing sea urchin histone genes (pSp 102), Drosophila ADH genes (sAC-1), and SV40 (SV2 CAT) replicate during the development of the injected frog embryo. In contrast, pBR322 either as monomers or multimers does not appear to replicate as efficiently. Generally, injected circular DNAs were not detectable by the gastrula stage of development, although there were several examples in which these molecules persisted until larval stages. In 90% of the cases, injected linear DNAs persisted as discrete molecules into early embryonic stages. A portion of the DNA sequences complementary to injected linear and circular molecules was detected comigrating with the high-molecular-weight cellular frog DNA (48 kb or larger) from mid-cleavage stages onward. Restriction enzyme analysis of DNA from injected embryos suggested some copies of the injected DNAs were integrated into the frog genome. This occurred in about 10%-30% of the cases of injected circular DNA and approximately 60%-70% of the cases of injected linear DNA. We were able to rescue circular plasmids from the injected blastulae by retransforming Escherichia coli. Restriction enzyme analysis of this DNA suggested that the majority of injected circular DNAs were not modified following replication in the frog embryo. The DNA of Xenopus embryos was highly methylated. On the other hand, injected DNA sequences were not methylated de novo even after many replication cycles in the frog embryo. Ribonucleic acid (RNA) transcripts from the injected DNAs were detectable by the late blastula stage of development.