The plasmids pEPI, peGFP, pdsRED, pOct4-eGFP (GOF18), and pγAChR were amplified in XL10 or ER2925 bacteria, isolated with anion exchange columns and resuspended in water. The DNA concentration was determined by a NanoDrop photometer; purity and supercoiled ccc conformation were verified by gel electrophoresis and suitable restriction endonucleases were employed to confirm identity of the plasmids. The dsRED-BAC (17) was grown in BH101 bacteria (provided by Frank Buchholz, Max Planck Institute for Molecular Cell Biology, Dresden, Germany) and purified using a commercial kit for large constructs (Large Construct Kit, Cat. no. 12412; Qiagen, Hilden, Germany). For BAC injections, the injection buffer was supplemented with 10 mM spermine and 23.3 mM spermidine (Sigma-Aldrich, Schnelldorf, Germany), which is 1/3 the polyamine concentration recommended for pronuclear injection of yeast artificial chromosomes (18).Minicircle preparation
Minicircles were produced essentially as described (10). A colony of Escherichia coli MM294Flp (provided by Francis Stewart, Biotechnologisches Zentrum der TU Dresden, Dresden, Germany) was transformed with a pEPI-type “parental plasmid” and grown overnight. Cells were collected and re-suspended in fresh Luria Bertani (LB) medium. Flp expression was initiated by incubation at elevated temperatures and resulted in the excision of prokaryotic plasmid parts, which, in the parental plasmid, are flanked by Flp-recombinase target (FRT) sites.In vitro methylation of plasmids
For methylation of cytosine bases at the 5 position (5mC) of CpG dinucleotides, a recombinant methyltransferase (SssI; New England BioLabs, Frankfurt, Germany) was used. Five milligrams of plasmid were incubated with 5 U SssI in the presence of 160 µM S-adenosyl-methionin. Complete methylation was verified by analytical restriction digests with the isoschizomers HpaII and MspI, respectively.Detection of 5mC by bisulfite sequencing
Bisulfite sequencing was essentially done as described (19). In brief, embryos were thoroughly washed in PBS, pools of 3–4 embryos were digested with proteinase K, and then DNA was precipitated. The resolubilized DNA was digested with EcoRI (New England BioLabs), denatured, and embedded in 7 µL 20 mg/mL low–melting point agarose (Biozym Scientific, Hessisch Oldendorf, Germany), cooled to form agarose beads, and incubated in 2.5 M bisulfite-hydroquinone solution pH 5 (Roth, Hamburg, Germany) for 4 h at 50°C. PCR was carried out in a final volume of 100 µL with individual agarose beads. The PCR fragments were separated by gel electrophoresis, isolated, and used for direct sequencing. Only sequences with >95% cytosine conversion of non-CpGs were analyzed. Methylation status of CpG dinucleotides was determined based on the ratio of cytosines to converted cytosines. The following primers were used to amplify the bisulfite-converted proximal Oct4 promoter: oct4bi1 (5′-GAT ATG GGT TGA AAT ATT GG-3′) and oct4bi2 (5′-AAA CTA AAC CCA ATC CAA CC-3′).Plasmid rescue after embryo in vitro development
Total DNA was isolated from peGFP-injected bovine embryos (n = 10), which showed eGFP fluorescence at blastocyst stages (day 8 of in vitro culture), and aliquots were used to transform highly electrocompetent ER1821 bacteria (Qiagen), which are lacking E. coli restriction systems. The transformation mixtures were then spread on LB agar plates containing kanamycin (50 µg/mL). Bacterial colonies arising after overnight culture at 37°C were employed for plasmid isolation and characterized by endonuclease restriction of the plasmids.Results Cytoplasmic injection of ccc plasmids
Here, we tested cytoplasmic injection of plasmids encoding fluorescent marker proteins into bovine and murine zygotes (Figure 1B). In transparent murine zygotes, the foreign DNA was deposited in the cytoplasm. Opaque bovine zygotes were not centrifuged, the cell membrane was penetrated by a microcapillary, and the DNA was injected into the cytoplasm. A dose-response test (not shown) revealed that the optimal plasmid concentration for cytoplasmic DNA injection was 10 ng/µL. After injection of ~10 pL plasmid solution [i.e., 100 fg (10-13 g) plasmid/zygote], the majority of zygotes (>80%) appeared morphologically intact and were placed in culture. Apparently, at this early stage, neither the presence of the S/MAR sequence nor the plasmid size had any overt effect on gene expression, as S/MAR-containing plasmids (pEPI and minicircle), conventional plasmids, and bacterial artificial chromosomes (BACs) were transcribed with equal efficiency (Figure 2, Table 1). On average, 50–60% of the bovine blastocysts expressed the fluorescent marker genes; approximately 50% of these showed dissimilar fluorescent intensities between blastomeres, suggesting mosaic or variegated transgene expression.