Aberrant DNA methylation in porcine in vitro‐, parthenogenetic‐, and somatic cell nuclear transfer‐produced blastocysts

Abstract

Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos. The relative methylation in the gamete and blastocyst samples showed that 18.5% (921/4,992) of the DNA clones were found to be significantly different (P < 0.01) in at least one of the samples. Furthermore, for the different blastocyst groups, the methylation profile of the in vitro-produced blastocysts was less similar to the in vivo-produced blastocysts as compared to the parthenogenetic-and somatic cell nuclear transfer (SCNT)-produced blastocysts. The microarray results were validated by using bisulfite sequencing for 12 of the genomic regions in liver, sperm, and in vivoproduced blastocysts. These results suggest that a generalized change in global methylation is not responsible for the low developmental potential of blastocysts produced by using in vitro techniques. Instead, the appropriate methylation of a relatively small number of genomic regions in the early embryo may enable early development to occur. Mol. Reprod.

Correlation of Developmental Differences of Nuclear Transfer Embryos Cells to the Methylation Profiles of Nuclear Transfer Donor Cells in Swine

“…Porcine DMH was conducted as previously described. 20 Porcine CpG island clones from a porcine CpG island library (PCGIL) (United Kingdom Human Genome Mapping Project, Hinxton, Cambridge, UK) were cultured in 96 well plates. The cloned inserts were amplified by polymerase chain reaction (PCR) using the library specific primers.…”

“…The PCR products were labeled with amino allyl-dUTP using the BioPrime labeling system as previously described, 20 with modifications. The PCR products were purified and resuspended in 29 ml H 2 O, mixed with 1X Bioprime buffer, dNTPs, Klenow and incubated for 60 minutes at 37°C.…”

“…Donor cell methylation profiles and bisulfite sequencing results were compared to the liver and in vivo-produced blastocyst methylation profiles and bisulfite sequencing results. 20 Bisulfite sequencing analysis. The DNA from the clonal donor cells was treated with bisulfite by using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, California) according to the vendor's recommendations.…”

“…Spots previously identified as differentially methylated in the donor cells and the blastocyst groups were selected for additional analysis by using bisulfite modified PCR. 20 The methylation status for five bacterial clones (B G 2 , HH A7, K D3, S E3 and X G 2 ) was determined in the liver or the clonal cell lines A2 or A7 (Fig. S1).…”

“…The methylation profiles of these donor cells were determined by using porcine differential methylation hybridization (PDMH) microarrays. 20 The methylation profiles were then correlated to the developmental potential of the cells.…”