2Helmholtz Zentrum für Infektionsforschung, Epigenetische Regulation, Braunschweig, Germany
K.I.'s current affiliation is the Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA, USA.
J.B.'s current affiliation is the Hannover Medical School, Hannover, Germany.
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). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter–controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.
A nonviral episomal plasmid system (pEPI) has been shown to be stably maintained in mammalian cells in vitro (1,2,3). Replication and mitotic stability of the pEPI plasmid are ensured by the presence of a human scaffold/matrix attachment region (S/MAR), which is located within a transcriptionally active cassette (3). The pEPI plasmid has been shown to replicate autonomously in several cells lines and primary cells and to be stably maintained as episomal entity in vitro (4,5,6). The generation of transgenic pig fetuses with the pEPI plasmid by sperm-mediated gene transfer has been reported (7). Minicircle vectors, which by definition are devoid of bacterial plasmid sequences, circumvent the defense mechanisms of mammalian cells, and resulted in persistent and high level expression of human factor IX and α1-antitrypsin in transfected mouse livers (8,9). Recently, episomal plasmids and minicircles could be combined to the first self-replicating nonviral minicircle (with S/MAR) that was stably expressed in the absence of selection pressure (10).
Here, we tested the suitability of S/MAR containing episomal vectors (pEPI and minicircle) and of conventional plasmids without S/MAR regions for ectopic gene expression in embryos by a simplified microinjection method. We chose the microinjection method, as this allows a precisely controlled application of foreign DNA. The classical method is injection of linearized DNA constructs into the pronuclei of fertilized eggs (Figure 1A) (11,12,13). However, successful injection of foreign DNA into the pronucleus requires high technical skills and expensive micromanipulation equipment, and is tainted with a high ratio of lysed zygotes, due to the particular vulnerability of the pronuclei (14). In addition, zygotes of some mammalian species, such as cow, contain high levels of colored lipids, which disguise the pronuclei. To visualize pronuclei in fertilized bovine oocytes, the zygotes are commonly centrifuged at 15,000× g (15), which may contribute to a reduced developmental capacity.
We show that cytoplasmic injection of supercoiled ccc plasmids into murine and bovine zygotes results in a high proportion of plasmid-expressing blastocysts. At this stage, the plasmids are episomally stable and promoter specificity of plasmid-encoded genes is maintained. Importantly, the onset of transcription of plasmid-encoded genes under control of the germ line–specific Oct4 (Pou5f1) promoter accurately reflects the species-specific time points of major embryonic genome activation and can be modulated by in vitro DNA methylation. The cytoplasmic plasmid injection into mammalian zygotes provides a simple and robust technique to experimentally access reprogramming events in early mammalian embryos.Materials and methods Ethics statement
Animals were maintained and handled according to German animal welfare guidelines.Generation of bovine and murine zygotes and microinjection of plasmids
Bovine oocytes were produced in vitro from slaughterhouse ovaries as described (16). For the isolation of murine zygotes, female NMRI mice (Harlan Winkelmann, Borchen, Germany) were superovulated, then caged with NMRI males for mating. Plugged females were sacrificed at day 0.5, and zygotes were isolated.
Plasmids were prepared in 10 mM Tris-HCl pH 8.0 and 0.25 mM EDTA, at a final concentration of 10 ng/µL and backfilled in glass injection capillaries. Individual zygotes were fixed by suction to a holding pipet (manufactured on a micro-forge in the laboratory; Reference 14), while the injection capillary was pushed though the zona pellucida and cell membrane. Approximately 10 pL plasmid solution was then injected into the cytoplasm using an Eppendorf transjector 5246 (Eppendorf, Hamburg, Germany).
Bovine embryos were cultured in groups of five in microdrops of synthetic oviductal fluid (SOF; Reference 16) medium under silicone oil with reduced oxygen tension (5%) at 39°C for 8 days. Murine embryos were cultured in potassium simplex optimized medium (KSOM) at 37°C for 4 days. Developing embryos were monitored daily to determine onset of fluorescent protein expression using a Zeiss Axiovert 35M microscope equipped with fluorescence optics (Zeiss, Göttingen, Germany; Reference 16) for Hoechst 33342, enhanced GFP (eGFP), and rhodamine. Images were recorded on Ektachrome 320 T films (Kodak, Stuttgart, Germany) using fixed exposure times of 10 s and 30 s for the normalization of eGFP and dsRED fluorescence intensities. For some specimens, a LSM confocal microscope (Zeiss) was used.