Episomal minicircles persist periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remain episomal and are not integrated into the genome. Minicircles survive extended periods without transcription in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of nonviral S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircle for the generation of transgenic animals.