Injection of murine zygotes with supercoiled plasmids or minicircles was less successful. About 80% of the treated zygotes cleaved; however, only 20–30% of the blastocysts expressed eGFP (Table 1). The larger volume of a bovine zygote may potentially facilitate DNA injections. We cannot exclude that in some cases, the plasmids may be unintentionally injected into a pronucleus, but this seems to be unlikely for the majority of embryos since the injections were performed at rapid frequency.
The relatively high success rates with cytoplasmic injection prompted us to initiate co-injection of two different plasmids (Figure 2E). The plasmids, peGFP (encoding eGFP) and pdsRED (encoding Discosoma red fluorescent protein), were mixed in equimolar ratios and co-injected into 77 bovine zygotes, resulting in a total of 18 blastocysts. Out of these, 8 blastocysts showed co-expression of eGFP and dsRED in almost all blastomeres (Figure 2E). No embryos expressing only one marker could be found. This suggests that the success of injection of a plasmid encoding a functional transgene of choice can be easily monitored by co-injection with a fluorescence marker–encoding plasmid.Reprogramming of ccc plasmid–encoded foreign DNA
Next we determined whether the observed expression patterns reflect the promoter-specificity of plasmid-encoded sequence, or whether the supercoiled plasmids become transcribed by a default mechanism. Plasmids with three different promoter sequences driving an eGFP open reading frame were injected into the cytoplasm of bovine zygotes, respectively. The cytomegalovirus (CMV) immediate-early promoter is ubiquitously active in cell culture, the murine Oct4 promoter has been proven to be germ line–specific (20,21) and the acetylcholine receptor γ-subunit (γAChR) promoter is exclusively active in skeletal muscle (22,23). Both the ubiquitous CMV promoter and the germ line–specific Oct4 promoter directed active transcription in injected embryos (Table 2), albeit with different time points of expression onset. The CMV promoter–controlled expression started from early 4-cell stage in bovine embryos. The γAChR promoter did not give rise to detectable eGFP fluorescence (Table 2). In the case of the γAChR promoter, no eGFP-positive blastomere could be detected in blastocysts or embryos degenerated at earlier stages.
The expression patterns of the Oct4-eGFP plasmid were investigated in greater detail during preimplantation development of bovine and murine embryos. The Oct4-eGFP plasmid was either injected unmodified or in a DNA-methylated version, which carried cytosine methylation (5mC) at CpG dyads (Figure 3). The unmodified Oct4-eGFP (25 injected mouse zygotes) evoked immediate expression of eGFP in late one-cell stage mouse embryos (i.e., ~12 h post-injection), whereas bovine embryos (50 injected zygotes) showed eGFP fluorescence only from the 4–8 cell stage onwards (i.e., 50 h post injection) (summarized data in Figure 3A). Importantly, the species-specific onset of eGFP transcription coincide well with major embryonic genome activation in these species, which starts at the late one cell stage in mouse and in 8-cell stage in bovine embryos (24,25).
In contrast, cytoplasmic injection of methylated Oct4-eGFP delayed the onset of eGFP expression to the late 8-cell stage, both in murine (n = 33) and bovine embryos (n = 63). Oct4 promoter activity has been studied in detail in the mouse; in transgenic mice carrying the Oct4-eGFP construct (20) expression of embryonic Oct4-eGFP becomes first detectable in 8-cell stages (26). Thus the 5mCpG-methylated Oct4-eGFP plasmid accurately reflected transcription of Oct4 from the embryonic genome.
To determine whether the time point of injection influenced the onset of expression, unmethylated (n = 49) and methylated Oct4-eGFP (n = 63) plasmids were injected in bovine metaphase II oocytes, respectively, followed by in vitro fertilization and culture. However, the onset of eGFP expression as detected by fluorescence (not shown) was indistinguishable from results obtained after zygote injections.