It has been postulated that active transcription of the Oct4 promoter (26,27,28) requires DNA demethylation and that DNA methylation results in transcriptional silencing of the Oct4 promoter. The results obtained herein with the CpG-methylated Oct4-eGFP plasmid (Figure 3) seem to support this notion. To provide further evidence, 5mCpG methylated Oct4-eGFP was injected into bovine zygotes and eGFP-positive morulae were collected after 6 days of culture. After thoroughly washing the embryos, total DNA was isolated and analyzed by bisulfite sequencing to identify the methylation status of the introduced Oct4 promoter sequences. Unexpectedly, all analyzed samples turned were fully methylated at CpG sites of the proximal murine Oct4 promoter (Figure 4).
Bisulfite sequence reads also reproducibly identified 5mC in two non-CpG sites (indicated by stars in Figure 4). A more detailed analysis revealed that these 5mC sites were positioned in pentameric recognition sequences of the bacterial Dcm methylase (consensus sequence: CCWGG), suggesting that the bacterial methylation marks of the XL-10 E. coli strain used for plasmid amplification were maintained. This finding strongly suggests that the Oct4-eGFP plasmids did not replicate and did not integrate into the genome, but indeed were episomally maintained. Non-CpG methylation has been reported previously in genomic sequences of eukaryotic cells; however, this was restricted to cultured embryonic stem (29) or lymphoma (30) cell lines, or to mouse oocytes before fertilization (31). Here, the injection of zygotes with unmethylated plasmids yielded bisulfite sequences, which were unmethylated at CpG dinucleotids and methylated at Dcm sites (data not shown), supporting the notion that the methylation changes of the embryonic genome do not affect the injected plasmids.
Further evidence for episomal maintenance of the injected plasmids was obtained by plasmid rescue experiments of bovine blastocyst stages (n = 10) at day 8 of in vitro culture. Electroporation of total DNA isolated from eGFP-positive cattle blastocysts into ER1821 bacteria resulted in 22 E.coli colonies resistant to kanamycin. Restriction analysis of the plasmids from 4 colonies showed that they were indistinguishable from the original plasmid (not shown). This suggests the majority of plasmids are episomally maintained and that no degradation of plasmids occurred during the culture period.Discussion
Here, we describe the development and validation of a modified DNA microinjection technique for the production of mammalian embryos with ectopic gene expression. The injection of ccc plasmids into the cytoplasm of zygotes from two mammalian species, mouse and cattle, turned out to be a simple, robust, and reliable technique to induce transcription of foreign DNA and expression of fluorescent marker genes. Preservation of the bacterial Dcm methylase–specific methylation pattern supports the notion that the majority of injected plasmids are maintained as episomal DNA. Nevertheless, the transcription of CMV promoter– and Oct4 promoter–controlled genes followed the species-specific embryonic genome activation time points (24,25). DNA methylated plasmids showed a delayed onset of transcription, suggesting that this epigenetic mark influences plasmid expression in blastomeres.
The rapid onset of expression of the unmethylated Oct4-eGFP plasmids in mouse embryos suggested that translocation of the plasmids into the nucleus happens in <12 h. How the plasmid DNA translocates into the nucleus after cytoplasmic injection is currently unclear. After nuclear membrane disassembly in the first cell cycle, some plasmid molecules might be taken up in the newly forming nucleus. A potential mechanism could involve the SV40 unit that has been found to facilitate nuclear translocation by association of ubiquitous transcription factors (10). The peGFP and the pEPI plasmids carry an SV40 promoter, which drives a kanamycin/neomycin selection cassette. In the minicircle, the SV40 promoter drives eGFP.
Plasmids and BACs seemed to be equally effective to evoke marker gene expression in embryos when equal amounts of 100 fg DNA were injected, although this corresponds to 20,000 plasmid molecules for a 4.7-kb plasmid and 500 molecules for the dsRed-BAC (~210 kb). The large genomic sequences of the BAC somehow seem to facilitate translocation to nucleus and promote expression. The ccc conformation of the injected plasmids may help to stabilize them in the embryos. Due to their superhelical status, ccc constructs might be better transcriptional templates than linear DNA (32).