Classic experiments showed that pronuclear injection is one route for the production of transgenic mammals (11,12). In these early experiments, fetuses or offspring were genotyped by Southern blotting to detect the presence of foreign DNA. This procedure required integration of foreign DNA and amplification during the subsequent cell divisions. Direct comparison of several factors affecting the efficiency of microinjection showed that cytoplasmic injection of linear or circular constructs resulted in an extremely low integration frequency of 0.9%, compared with up to 33% for pronuclear injections of mouse zygotes (13). Episomal DNA plasmids, which do not replicate, would have been diluted to such an extent in fetuses or offspring that their concentration would be below the detection limit of Southern blotting. However, in early embryos with a total cell numbers of ~60 for murine and ~120 for bovine blastocysts, the number of plasmid molecules is apparently sufficient for strong ectopic gene expression.
The reason for the different efficiencies of plasmid-encoded expression in bovine and murine embryos may be due to the larger volume of a bovine zygote (diameter ~140 µm, volume ~1.44 × 106 µm3) compared with a mouse zygote (diameter ~80 µm, volume ~0.27 × 106 µm3). Compared with previous experiments in this laboratory with pronuclear injection of a linear Oct4-eGFP construct (21), the cytoplasmic injection was significantly more efficient. Cytoplasmic injection resulted in 40–60% eGFP-positive bovine embryos versus 4% for pronuclear injections in this species. Previously, intracytoplasmic injection (ICSI) of DNA-loaded sperm cells in metaphase II oocytes of mice (33), monkey (34), and cattle (35) has been described. With the exception of mouse embryos, cytoplasmic ccc plasmid injection seems to be significantly more efficient for the production of blastocysts with ectopic gene expression.
Unexpected findings were that (i) the transcription of the injected plasmids reflects the species-specific time point of major embryonic genome activation and (ii) the onset of transcription could be modulated by DNA methylation. The unmodified Oct4-eGFP evoked immediate expression of eGFP in late one-cell stage mouse embryos (i.e., ~12 h post-injection), whereas the unmodified Oct4-eGFP induced eGFP expression in bovine embryos from the 4–8 cell stage (~50 h post-injection) onwards. In contrast, the CpG-methylated Oct4-eGFP plasmid delayed onset of eGFP expression to late 8-cell stage, both in murine and bovine embryos. The genomic oct4 gene shows a biphasic expression during preimplantation development (36). In a study dissecting maternal and embryonic expression, the embryonic Oct4-eGFP was first detectable in 8-cell stages of mouse (26). In cloned Oct4-eGFP transgenic cattle embryos, the first eGFP expression could be detected just after the 8-cell stage (37). This strongly suggests that the plasmids are properly processed by the cellular machinery. The unmodified Oct4-eGFP plasmid behaved like the majority of embryonic genes, whereas the methylated Oct4-eGFP evoked a gene-specific expression pattern highly similar to endogenous or transgenic oct4 expression (19,26,36,37). In bovine embryos, it has been shown previously that injected DNA does not seem to be expressed <50 h post-fertilization using a β-actin promoter–lacZ construct (38); however, expression could also be detected in blocked 2-cell stages, when stained at the end of the culture. Correspondingly, we found that at day 7–8 of bovine embryo culture several of the blocked embryos (1-cell, 2-cell, 4-cell, or irregular cleavage stages) showed eGFP fluorescence. This highlights the need to analyze the onset of transgene expression at the appropriate time point of culture. Since livestock embryos are highly sensitive to temperature changes, the number of fully developed bovine and murine blastocysts might have been reduced by daily inspection under a fluorescence microscope. Total amount of injected DNA also seems important, as several of the blocked embryos showed unusually intense fluorescence.
At the moment it is not known where the plasmids are located in the embryonic nuclei or whether they are associated with histone proteins. In cell culture, the S/MAR region is assumed to be critical for episomal plasmid localization at the nuclear matrix and replication during cell divisions (1,2,3).
Importantly, for a ccc episome, demethylation does not seem to be necessary for transcription. This finding may have implications for the interpretation of genomic methylation patterns. It is unclear, however, why the introduced plasmids do not become demethylated up to the morula/blastocyst stage, as both the maternal and paternal gamete genomes of murine (39) and bovine embryos (40) undergo global demethylation starting after fertilization. In embryos cloned by somatic nuclear transfer, a reduced demethylation of the introduced genome has been detected (40,41).
In summary, cytoplasmic injection of supercoiled DNAs is a reliable, robust, and efficient method for ectopic gene expression in embryos and seems to be an ideal tool to study reprogramming events during early ontogenesis. Whether episomal plasmids with replication properties in mammalian embryos can be developed will require further research.
We thank Joseph W. Carnwath for helpful discussions and gratefully acknowledge the excellent technical help by Karin Korsawe, Erika Lemme, and Stephanie Holler. E. Lemme generously provided the image of Figure 1A. The pEPI plasmid was provided by Ina Stehle and Hansjörg Lipps (Universität Witten, Witten, Germany), Frank Buchholz (MPI Dresden) provided the dsRED-recombineered BAC clone, and Pessah Yamplonsky and Veit Witzemann (Max Planck Institute for Biomedical Research, Heidelberg, Germany) provided the γAChR plasmid. This work is part of the Ph.D. thesis of K.I. within the Ph.D. program of the Hannover Medical School (Hannover, Germany). K.I. was supported by a studentship from Goyaike, Buenos Aires. This work was funded in part by the Deutsche Forschungsgemeinschaft (DFG). Work by J.B. and S.B. was supported by the Excellence Initiative “REBIRTH” (from Regenerative Biology to Reconstructive Therapy), the SFB 738 (Optimierung konventioneller und innovativer Transplantate), and the CliniGene Network of Excellence (European Commission FP6 Research Program, contract LSHB-CT-2006-018933.
Competing interest statement
The authors declare no competing interests.
Correspondence Address correspondence to Wilfried A. Kues, Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Biotechnology, Mariensee, D-31535 Neustadt, Germany. email: [email protected]References
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