Cloning Stem Cells最新文献

Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog transcripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were methylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic enrichment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the parameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.

We determined the effect of heat shock (HS) on the alterations of development and calcium releasing capacity of nuclear-ooplasmic reconstructed porcine oocytes stimulated by thimerosal. The non-HS (39 degrees C) and the HS2h (41.5 degrees C for 2 h) matured oocytes were enucleated and their spindles/chromosomes were exchanged between these two groups followed by parthenogenetic activation. In the Control group (Csp-Coop), the non-HS spindle (Csp) was transferred to the non-HS ooplasm (Coop). Blastocyst and cleavage rates were higher in both Csp-HSoop (non-HS spindle transferred to the HS ooplasm) and HSsp-Coop (HS spindle transferred to non-HS ooplasm) reconstructed oocytes, but no difference was detected in the average cell number per blastocyst. However, intracellular calcium concentrations ([Ca(2+)](i)) generally declined (p < 0.05) in the reconstructed HS oocytes, with a greater blastocyst rate after parthenogenetic activation. In the present study, time for the completion of spindle transfer in these oocytes was 1-2 h, during which some physiological remodeling or adaptation might have been occurred in the oocytes. Therefore, changes in heat-shock protein70 (HSP70) expression and developmental competence of the HS2h oocytes with 1 or 2 h of recovery time under normal culture temperature (39 degrees C) were examined. The results showed that the expression of HSP70 in the HS2h oocytes was higher (p < 0.05) than those had recovery incubation for 1 h (HC1h) after HS, but the cleavage and blastocyst rates were greater (p < 0.05) in the HC1h group. We demonstrated that a recovery period prior to activation of porcine oocytes and reconstructed oocytes is beneficial to further development. Heat shock to either the karyoplast or the ooplasm enhances embryonic development but reduces intracellular calcium release in the cloned porcine oocytes.