305 REPROGRAMMING EVENTS IN EARLY BOVINE AND MURINE EMBRYOS ARE MIRRORED BY PLASMID-ENCODED MARKER CONSTRUCTS

Abstract

Episomal plasmids have emerged as useful tools to achieve stable transgenesis in mammalian cell cultures. Here, the suitability of scaffold/matrix attachment region (S/MAR) carrying episomal plasmids and conventional plasmids for the generation of transgenic murine and bovine embryos was assessed. Bovine zygotes were produced from slaughterhouse ovaries, and murine zygotes were isolated from superovulated and mated NMRI females. Zygote stages were microinjected with approximately 10 pl of plasmid solution. The S/MAR encoding plasmids pEPI or minicircle preparations (gift of J. Bode, Braunschweig, Germany), devoid of most of the plasmid backbone, were used as episomal plasmids. Both plasmids carry an enhanced green fluorescent protein (EGFP) gene driven by the cytomegalovirus promoter (CMV). The plasmids peGFP (CMV-eGFP), pdsRED encoding red fluorescent protein (CMV-RFP), pOct4-GFP (germ line-specific Oct-4 promoter-EGFP), and pgAChR-GFP (muscle-specific γAChR promoter) were used as conventional plasmids. To study the effects of DNA methylation at cytosine/guanine dinucleotids (CpG), plasmid DNA was treated with CpG-methylase in the presence of S-adenosyl-methionin, and in some experiments, completeness of DNA methylation was verified by methylation-sensitive restriction endonucleases. Embryos were analyzed during in vitro culture up to blastocyst stage by fluorescence microscopy, and selected stages were harvested for RT-PCR analysis or DNA recovery. Microinjection of circular plasmids with ubiquitous CMV (n = 505) or germ line-specific Oct-4 promoter (n = 176) driven transcription in bovine zygotes resulted in 159 and 44 blastocysts, of which 94 and 27 showed expression of EGFP. Microinjection of bovine zyotes (n = 179) with S/MAR plasmids yielded a total of 18 blastocysts of which 12 were green fluorescent protein-positive. On average, >50% of the blastocysts were EGFP-positive, irrespective of whether S/MAR carrying episomal plasmids or conventional plasmids had been injected. In contrast, injection of the γAChR (muscle-specific) driven construct did not give rise to EGFP expression (n = 20), suggesting that promoter specificity was maintained. Injection of murine zygotes (n = 126) with CMV or Oct-4 promoter constructs was less successful, about 10 to 20% of the obtained blastocysts expressed EGFP. In the case of unmethylated pOct4-GFP plasmid, the onset of EGFP expression was found to coincide with the time point of major embryonic genome activation [i.e. late 1-cell stage in murine (n = 25) and 4- to 8-cell stages in bovine (n = 75) embryos]. In contrast, injection of CpG-methylated plasmids (murine n = 33; bovine n = 101) delayed the onset of EGFP expression for a further 30 to 40 h. Recovery of plasmid sequences from blastocyst stages and bisulfite sequencing indicated that the majority of plasmids are maintained in an episomal status. Thus, plasmid-mediated transgenesis is a robust method to express foreign DNA in a promoter-specific manner in mammalian embryos and can be employed to analyze reprogramming events.

The excellent technical support by E. Lemme and K. Korsawe is acknowledged. Funded by DFG.