PCR detection of Oct4 mRNA in SCNT and ICSI embryos (C3HxB6).

Pronuclear stage oocytes at 6 hours post activation (hpa) following SCNT were cultured in 2 µg/mL aphidicolin (aph) or in 5 µg/mL cytochalasin B (CB) until 48, 60 and 72–96 hpa to test the requirement of the first round of DNA replication and cell division for reprogramming. As controls, pronuclear stage oocytes were treated the same way after ICSI, or were cultured in normal medium and allowed to cleave to 2-cell, 4-cell and morula-blastocyst stage. To test the requirement of the second round of DNA replication and cell division for reprogramming, 2-cell embryos at 24 hpa were cultured in 2 µg/mL aph or in 5 µg/mL CB until 60 hpa and were compared with time-matched 8-cell embryos. As controls, 2-cell embryos were treated the same way after ICSI. For each treatment and type of embryo, results are given as ratio of frequencies, as follows: embryos expressing C57Bl/6J Oct4 mRNA/embryos expressing Oct4 mRNA/embryos positive for β-actin mRNA. Statistical analysis was conducted on the ratio C57Bl/6J Oct4/β-actin mRNA using chi-square test and applying the Bonferroni correction for multiple comparisons.a,b: different superscripts within same row indicate significant difference (chi-square test).n.p.: not performed.total Oct4: Oct4 from maternal, paternal/somatic, or bi-allelic origins.