Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos
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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 remained episomal and were not integrated into the genome. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of 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 minicircles for the generation of transgenic animals.
KeywordsScaffold/matrix attachment region Minicircle Episome Transgenic animals Plasmid
We would like to thank Fleur Oback and Jan Oliver for assistance in SCNT, Jaime Oswald for outgrowing cells from blastocysts and Pauline Hunt for assembling and preparing the figures for this manuscript.
This work was supported by the Ministry of Business, Innovation and Employment (AgResearch Core Fund A16305, A13654 and A19064).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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