We prepared sperm nuclei and egg S-phase extract as described elsewhere Fluorescence in situ hybridization FISH was performed as described elsewhere 67 using the major satellite probe of bp that contained two bp units. Since the early s a small number of notable experiments have provided significant impetus to the field, primarily the demonstration of reprogramming ability, first by the complex cytoplasmic milieu that constitutes the amphibian egg, then that of the mammalian egg, and finally that of the mammalian embryonic stem cell. Nuclear Reprogramming and Stem Cells will serve as a valuable resource for all researchers in the field of stem cell biology, including those just setting out on their career path as well as those already established in the field. The lack of obvious nuclear swelling in these experiments suggested that chromatin decondensation was not a secondary effect of nuclear swelling. From our study based on the hypothesis that global decondensation of somatic cell chromatin in egg cytoplasm may facilitate reprogramming of the somatic cell genome, we found that Npm could decondense both euchromatin and centromeric heterochromatin, primarily in undifferentiated mouse nuclei. Due to the highly condensed heterochromatin, mouse centromeres are clearly visible as DNA-dye-positive spots in interphase cells by using fluorescence microscopes. Later, it was found that during sperm chromatin decondensation upon fertilization, Npm replaces sperm-specific basic proteins X and Y with egg histone H2A and H2B, resulting in assembly of somatic-type nucleosomes onto sperm DNA 59 , Importantly, in addition to offering the opportunity to delve deeply into the mechanisms that drive disease aetiology the research is realistically opening the doors for development of targeted and personalized therapeutic applications that many considered, until recently, to be nothing more that a far fetched dream. Egg Npm eNpm was immunodepleted from egg extract using polyclonal anti-Npm antibody as described previously The fractions that showed chromatin decondensation, eluted by to mM NaCl, were pooled, dialyzed against buffer C, and applied to HiTrap heparin.
This study provides new insight into the molecular and functional analyses of chromatin decondensation in the context of somatic cell nuclear cloning. I originally hail from Rome, Italy, where I grew up, matriculated, attended medical school, and received training in general and transplant surgery. Methods and Protocols, hands-on laboratory scientists describe readily reproducible methods for actually performing nuclear reprogramming by nuclear transfer in several different species, by fusion induced both chemically and electrically, and by the in vivo treatment of cells with cell extracts. Nucleus-wide chromatin decondensation might facilitate reprogramming of the donor nuclei by derepressing condensed chromatin; however, there is a wide knowledge gap between chromatin decondensation at the microscopic level and derepression at the transcriptional level. The supernatant and pellet were applied to two sodium dodecyl sulfate gels for staining with Coomassie brilliant blue R and immunoblotting, respectively. The extract could also decondense centromeric heterochromatin in the mouse embryonal carcinoma F9 cells, as demonstrated by using anti-tm-H3K9 antibody and the DNA dye Topro 3. My main achievements have been in the field of steroid-free immunosuppression IS , IS minimization and clinical tolerance after liver transplantation, as well as in the development of platforms for renal, small bowel and endocrine pancreas bioengineering and regeneration research. I have authored more than research papers, review articles, and book chapters. We recognize that research into many other disease states and cell types are all equally worthy of discussion. Methods and Protocols significantly expands the repertoire of methods available to perform, study, and understand nuclear programming, not only for cloning, but also for reprogramming somatic cells to stem cells. In contrast to the round centromeres observed in untreated F9 nuclei and the nuclei treated with buffer B for 2 h, the nuclei incubated with egg extract showed irregularly spread centromeres Fig. Electron microscopy was performed as described previously At the functional level, the chromatin decondensation facilitated new gene expression as shown by the nuclear transplantation into oocytes. We show that this decondensation was not accompanied by histone release from DNA, unlike in sperm chromatin decondensation, but it was accompanied by a variety of epigenetic modifications. One of the key questions in nuclear cloning is whether a few general reprogramming factors exist that can nonspecifically affect multiple genes in addition to the obviously necessary gene-specific activators and suppressors. Nuclear swelling and chromatin decondensation in egg cytoplasm have mainly been studied in the more physiological context of sperm chromatin decondensation upon fertilization. The main goal of my investigations is to bio-engineer and implant in humans renal organoids, insulin-producing cells and segments of small bowel. The protocols follow the successful Methods in Molecular Biology series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. In our current work we applied the assay to study widespread chromatin decondensation in somatic nuclei incubated in Xenopus egg extract, expecting that the decondensation factor might be one of the general reprogramming factors described above. The supernatant was labeled as 0. Nuclear Reprogramming and Stem Cells will serve as a valuable resource for all researchers in the field of stem cell biology, including those just setting out on their career path as well as those already established in the field. The Npm monomer is composed of two domains, the N-terminal core domain containing amino acids and the C-terminal tail domain. The primary defects in the mNpm2 null mice were dispersed nucleoli and female infertility. The main goal of my scientific production is to orchestrate the transition of organ transplantation towards a regenerative medicine-focused type of research. Methods and Protocols Steve Pells 0 Recenzije Although nuclear reprogramming-the cellular process underlying cloning and the artificial generation of stem cells from somatic cells-may be considered a "black box" whose interior events are either completely unknown or at least imperfectly characterized, tantalizing clues about its operation are beginning to emerge. Xenopus Npm consists of or four residues deleted amino acids and forms a pentamer in vivo. Sources of the primary antibodies were as follows:
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