Table 3. PCR detection of Nanog mRNA in SCNT and ICSI embryos (C3HxB6).

	No. embryos Nanog+/β-actin+ (%)
Groups	Aph−	Aph+	CB+
48 hpa SCNT (treated at 6 hpa)	6/20 (30)a	1/17 (6)a	1/10 (10)a
48 hpa ICSI (treated at 6 hpa)	9/14 (64)a	1/14 (7)b	7/8 (88)a
60 hpa SCNT (treated at 6 hpa)	34/54 (63)a	0/27 (0)b	12/34 (35)b
60 hpa SCNT (treated at 24 hpa)	10/33 (30)a	2/38 (5)b	n.p.
60 hpa ICSI (treated at 6 hpa)	32/50 (64)a	0/24 (0)b	38/69 (55)a
60 hpa ICSI (treated at 24 hpa)	16/17 (94)a	26/45 (58)b	n.p.
72–96 hpa SCNT (treated at 6 hpa)	23/39 (59)a	1/28 (4)b	9/23 (39)a
72–96 hpa ICSI (treated at 6 hpa)	53/65 (82)a	2/40 (5)b	43/57 (75)a
Pooled SCNT (treated at 6 hpa)	63/113 (56)a	2/72 (3)b	22/67 (33)b
Pooled ICSI (treated at 6 hpa)	94/129 (73)a	3/78 (4)b	88/134 (66)a

Pronuclear 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 oocytes were treated the same way after fertilization, 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 fertilization. For each treatment and type of embryo, results are given as ratio of frequencies, as follows: embryos expressing Nanog mRNA/embryos positive for β-actin mRNA. Statistical analysis is conducted on the ratio Nanog/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.