Transcriptional failures arrest development after human nuclear transfer
ABSTRACT: Reprogramming occurs after nuclear transfer into zygotes whose genomes have been removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase. Our results suggest that there is a previously unappreciated barrier to successful human nuclear transfer, and that future studies should focus on the requirements for somatic genome activation. Overall design: 1-3 embryos were used for analysis. RNA amplification was done using two or three rounds of T7-mediated RNA amplification using the Illumina Total Prep RNA Amplification kit. Somatic cells 1-000 and 1-011 required only one round of RNA amplification because starting amounts of RNA were 100-500ng, while embryonic samples were amplified from single cells or embryos.
Project description:Reprogramming occurs after nuclear transfer into zygotes whose genomes have been removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase. Our results suggest that there is a previously unappreciated barrier to successful human nuclear transfer, and that future studies should focus on the requirements for somatic genome activation. 1-3 embryos were used for analysis. RNA amplification was done using two or three rounds of T7-mediated RNA amplification using the Illumina Total Prep RNA Amplification kit. Somatic cells 1-000 and 1-011 required only one round of RNA amplification because starting amounts of RNA were 100-500ng, while embryonic samples were amplified from single cells or embryos.
Project description:Reprogramming occurs after nuclear transfer into zygotes whose genome was removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase Egli et al. Development 2010 doi:10.1242/dev.046151 Groups of 20 mouse embryos were used for the analysis. RNA amplification was done using Illumina total prep RNA amplification kit. Total of 21 arrays.
Project description:Reprogramming occurs after nuclear transfer into zygotes whose genome was removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase Egli et al. Development 2010 doi:10.1242/dev.046151 Overall design: Groups of 20 mouse embryos were used for the analysis. RNA amplification was done using Illumina total prep RNA amplification kit. Total of 21 arrays.
Project description:It has been demonstrated previously that the reprogramming factors are sequestered in the pronuclei of zygote after fertilization, as the enucleated zygotes at interphase cannot support the development of cloned embryos whereas the enucleated zygotes at M-phase can reprogram somatic cells to full pluripotency. However, it remains unknown whether the parental pronucleus, derived either from the sperm or oocyte, possesses the similar reprogramming ability. Here, we provide evidence demonstrating that the parental pronuclei are asymmetric in reprogramming and the reprogramming factors reside mainly in the male pronucleus. As a result, only the female pronucleus-depleted mouse zygotes enucleated at M-phase of mitosis can support the somatic cell reprogramming, the derivation of chromosome transfer embryonic stem (ctES) cells with full pluripotency and the full term development of cloned embryos. In striking contrast, the male pronucleus-depleted zygotes enucleated at M-phase of mitosis fail to support the pre-implantation development of somatic cell cloned embryos. Furthermore, we demonstrated that the distinct epigenetic reprogramming ability of the parental pronucleus might contribute directly to the developmental difference of somatic cloned embryos. Our study highlights the developmental asymmetry of parental pronuclei in reprogramming. Overall design: We compared the gene expression profile of ctES cells. Two biological repeats were included.
Project description:Mammalian oocytes have the ability to reset the transcriptional program of differentiated somatic cells into that of totipotent embryos through somatic cell nuclear transfer (SCNT). However, the mechanisms underlying SCNT-mediated reprogramming are largely unknown. To understand the mechanisms governing chromatin reprogramming during SCNT, we profiled DNaseI hypersensitive sites (DHSs) in donor cumulus cells and 1-cell stage SCNT embryos. To our surprise, the chromatin accessibility landscape of the donor cells is drastically changed to recapitulate that of the in vitro fertilization (IVF)-derived zygotes within 12 hours. Interestingly, this DHS reprogramming takes place even in the presence of a DNA replication inhibitor, suggesting that SCNT-mediated DHS reprogramming is independent of DNA replication. Thus, the study not only reveals the rapid and drastic nature of the changes in chromatin accessibility through SCNT, but also provides a DNA replication-independent model for studying cellular reprogramming. Overall design: Here we performed low-input DNase-Seq (liDNase-Seq) profilling of Donor cumulus cells, 1-Cell stage somantic nuclear Transfer (SCNT) embryos and in 1-Cell Aphidicolin treated SCNT embryos
Project description:Enucleated oocytes have the remarkable ability to reprogram somatic nuclei back to totipotency. Here we investigate genome-scale DNA methylation patterns after nuclear transfer and identify specific targets for DNA demethylation as well as NT specific limitations. We find that the ooplasm can confer similar demethylation patterns in both processes except at some repetitive element classes and that germ-line associated promoters are specifically targeted in NT. Comparison of DNA methylation patterns in murine fertilization and somatic cell nuclear transfer
Project description:Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle. Overall design: 13 samples were analyzed. Aph-IVF: Aphidicolin not treated, in vitro fertilization, 1 biological rep Aph-IVF96: Aphidicolin not treated, in vitro fertilization (96h), 1 biological rep Aph-IVFBla: Aphidicolin not treated, in vitro fertilization blastocyst, 1 biological rep Aph+IVF: Aphidicolin treated, in vitro fertilization, 1 biological rep Aph+IVF96: Aphidicolin treated, in vitro fertilization (96h), 1 biological rep Aph+IVFBla: Aphidicolin treated, in vitro fertilization blastocyst, 1 biological rep Aph-SCNT: Aphidicolin not treated, somatic cell nuclear transfer, 1 biological rep Aph-SCNT96: Aphidicolin not treated, somatic cell nuclear transfer (96h), 1 biological rep Aph-SCNTBla: Aphidicolin not treated, somatic cell nuclear transfer blastocyst, 1 biological rep Aph+SCNTBla: Aphidicolin treated, somatic cell nuclear transfer blastocyst, 1 biological rep B6C3F1-Ooc: Aphidicolin not treated, B6C3F1 oocytes, 1 biological rep B6C3F1-Cum: Aphidicolin not treated, B6C3F1 cumulus cells, 2 biological rep
Project description:Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease. Overall design: RNA sequencing collected from PBNT1 and hESO-14
Project description:Genome wide comparison of gene expression between EpiSC lines derived from fertilized (FT) embryos and somatic cell nuclear transfer (NT) embryos. EpiSC lines were derived from fertilized and somatic cell nuclear transfer embryos and cultured until 15 to 20 passages. RNA was then extracted in order to compare transcriptomic profiles. Overall design: Total RNA was extracted from 3 FT-EpiSC lines, and 3 NT-EpiSC lines. For each line, there were 3 biological repeat.
Project description:The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells, holding promise for autologous cell replacement therapy. Though reprogramming of somatic cells by nuclear transfer was first demonstrated more than 60 years ago, only recently have human diploid embryonic stem cells been derived after nuclear transfer of fetal and neonatal fibroblasts. Because of the therapeutic potential of developing diploid embryonic stem cell lines from adult cells of normal and diseased human subjects, we have systematically investigated the parameters affecting efficiency and developmental potential in their derivation. We found that improvements to the oocyte activation protocol, including the use of both a kinase and a translation inhibitor, and cell culture in the presence of histone deacetylase inhibitors enable development of diploid cells to the blastocyst stage. Developmental efficiency varied significantly between oocyte donors, and was inversely related to the number of days of hormonal stimulation required to reach mature oocytes, while the daily dose of gonadotropin or the total number of MII oocytes retrieved did not affect developmental outcome. The use of diluted Sendai virus in calcium-free medium during nuclear transfer improved developmental potential, while the use of concentrated Sendai virus induced an increase in intracellular calcium and caused premature oocyte activation. Using these modifications to the nuclear transfer protocol, we successfully derived diploid pluripotent stem cell lines from both postnatal and adult somatic cells of a type 1 diabetic subject. The goal of this experiment was to determine if human oocytes have the ability to reprogram a somatic cell genome in the absence of the oocyte genome. Our previous research had indicated that human oocytes can reprogram adult somatic cells if the oocyte genome remains present (Noggle et al. Nature 2011, doi:10.1038/nature10397). The data presented here is part of a new series of experiments aimed at obtaining diploid cells after somatic cell nuclear transfer into enucleated oocytes. In this experiment, adult somatic cells were transferred into enucleated oocytes and thereafter cultured in the presence of 240ng/ml scriptaid for 17 hours. Samples were cultured until cleavage stage and then collected for microarray analysis.