Cytoplasmic injection of circular plasmids allows targeted expression in mammalian embryos.

Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs).

Minicircle performance depending on S/MAR-nuclear matrix interactions.

The ideal vector for cell and tissue modification does not depend on integration but rather behaves as an independent functional unit that replicates as an episome. Based on a scaffold/matrix attachment region (S/MAR), we have introduced, in 2006, an approximately 4-kb replicating nonviral minicircle able to exploit the cellular replication machinery in a way reminiscent of ARS vectors. Consisting of only one active transcription unit and the S/MAR, it resists silencing as it is free of prokaryotic vector parts and drug selection markers.