Fig. 4.

Incomplete establishment of imprinting at the hIC1 in knock-in male germ cells. (A) Percent methylation at IC1 measured by pyrosequencing; assays d and l were used (Fig. 1C). From left to right, results for endogenous hIC1 in mature sperm samples from fertile men (hSP1 and hSP2), the endogenous mIC1 and targeted hIC1 in knock-in sperm (KISP) from adult mice, and the targeted hIC1 in pools of H19^+/hIC1 two-cell stage embryos (KI2 cells) are shown. KISP, n = 6 mice; KI2 cell pools, n = 3. *P < 0.05, two-tailed Student's t test with equal variance. Bars represent mean ± SEM; error bar of the KISP (mIC1) is too small to be seen on the graph. (B) Methylation at hIC1 in KISP and hSP1 analyzed by bisulfite treatment followed by sequencing. Assays e–h were used (Fig. 1C). Cytosines in CG dinucleotides that are conserved between mouse and human and located within CTS are depicted as black lines above the clones and marked as CTS1, 2, 3, 4, and 6 (13). Cytosines measured by pyrosequencing are marked with an asterisk. (C) ChIP–qRT-PCR for H3K4me2 and H3K9me3 at mIC1 and hIC1 in knock-in round spermatids. Assays b and j were used (Fig. 1C). Two independent pools of round spermatids were generated as detailed in Materials and Methods, and the results from each pool are shown separately.