Kinetics of transcription and H3.3 incorporation following nuclear transplantation to Xenopus oocytes. (A) Schematic representation of the Oct4 transgene composed of a 20 copy array in the embryonic stem cells used in this study (ESC #5). DE, Distal enhancer; PE, Proximal enhancer; PP, Proximal promoter. (B) qRT-PCR analysis of transcription during a 48-h period following nuclear transplantation of 4 day-retinoic acid differentiated ESC #5 nuclei. Error bars indicate s.e.m. (n = 3). (C) Analysis of rDNA and major satellite transcription rates. Oocytes were transplanted with RA-ESCs and injected with BrUTP at various times after nuclear transfer (at 0, 12, 24, or 36 h). Oocytes were then collected 12 h after the time of BrUTP injection. In that way, the different samples collected will contain transcripts produced by the transplanted nuclei and labeled by BrUTP during a 12-h period (T12 to 24 h, T24 to 36 h, T36 to 48 h). After mRNA extraction BrUTP labeled transcripts are purified by immunoprecipitation with an anti-BrdUTP antibody and analyzed by qRT-PCR. Error bars indicate s.e.m. (n = 3). (D) ChIP analysis of histone incorporation 24 h after NT in oocytes preinjected with mRNA for HA-H3.3 and HA-H3.2, performed over the Oct4 reporter promoter, major satellite and ribosomal DNA genes, respectively. The total amount of incorporated H3.3 is set to 1. Error bars indicate standard deviation (n = 3). (E) Analysis of histone incorporation kinetics over Oct4 regulatory regions after NT. Four-day retinoic acid treated ESC #5 were transplanted in oocytes preinjected with mRNA for HA-H3.3 and HA-H3.2, collected over a 48-h time course and analyzed by ChIP with an HA antibody.