No description available.

The objective of this study was to investigate whether or not the treatment of nuclear donor cells with inhibitors of protein synthesis and cyclin-dependent kinases (CDKs) affect the development of swine embryos produced by somatic cell nuclear transfer. Host oocytes were derived from pre-pubertal females and matured in vitro for 42-46 h under standard conditions. Nuclear donor cells were fetal fibroblasts maintained in culture for 2 to 6 passages. Oocytes were reconstructed with cells treated for 22-24 hours with cycloheximide (CHX; 10mug/ml), roscovitine (ROS; 25 muM), the combination of CHX + ROS (CR), or untreated cells. Two hours after reconstruction, the oocytes were activated using ionomycin (15muM/5 min) and strontium chloride (10mM/4h), maintained for 6 h in the presence of cytochalasin B (7.5mug/ml) and CHX (10mug/ml), and then cultured in porcine zygote medium (PZM3) for 6 days. The cleavage rate, 63.7% (n=318), 55.2% (n=99), 56.7% (n=107) and 60.6% (n=347), at 48 h post-fusion were not significantly different between embryos derived from ROS, CHX, CR and control cells, respectively. Developmental rate to blastocyst stage was higher for embryos reconstructed with ROS (12.2%) and untreated cells (12.1%) when compared to CHX (5.7%) and CR (4.9%). Blastocysts produced with ROS treated cells had similar number of nuclei compared to embryos reconstructed with untreated donor cells (30.9+/-10.4 vs. 32.2+/-8.0). Phosphorylated H2A.X (gammaH2A.X) was highly expressed in donor cells treated with CR compared to non treated cells, but it was similarly expressed in most of 1-cell stage embryos reconstructed with control or treated cells. Flow cytometry analysis showed that the majority of the fibroblasts were at G 0/G1 phase of the cell cycle at the time of nuclear transfer. It was concluded that the treatment of nuclear donor cells with inhibitors of protein synthesis and CDKs did not improve the in vitro development of somatic cell nuclear transfer embryos in pigs.

No description available.

Polyglutamine (polyQ) diseases are a group of progressive neurodegenerative disorders, which are caused by the expansion of an existing glutamine-coding CAG repeat in the coding region of disease genes. The cell nucleus is a major site of polyQ toxicity, and gene transcription is compromised in polyQ-induced neurodegeneration. Understanding the nuclear translocation of mutant polyQ proteins is therefore crucial to unfold the complex pathogenic mechanisms that underlie the neuronal toxicity of polyQ disease. The polyQ domain is the only common sequence found among different mutant disease proteins. Nuclear transport signals have been identified in some, but not all, polyQ disease proteins. The detection of those mutant polyQ proteins that carry no classical nuclear transport signal, but not their normal counterparts, in the cell nucleus suggests the existence of uncharacterized nuclear transport signals in mutant polyQ proteins. Thus, the objective of the present study is to elucidate the nuclear transport pathway(s) adopted by an expanded polyQ domain and determine its correlation with polyQ toxicity. / Through a series of genetic and biochemical studies in cell culture, mouse and transgenic Drosophila models, exportin-1 was found to modulate the nucleocytoplasmic localization of mutant polyQ protein and its toxicity. Further, mutant polyQ protein was also demonstrated to be a novel transport substrate of exportin-1. By promoting the nuclear export of mutant polyQ protein, exportin-1 suppressed polyQ toxicity by reducing the interference of mutant polyQ protein on gene transcription. It was found that the protein level of exportin-1 diminished in the normal ageing process, which would result in an exaggeration of nuclear mutant polyQ toxicity. Thus, the age-dependent decline of exportin-1 level, at least in part, accounts for the progressive degeneration observed in polyQ patients. Results obtained from this project first demonstrated that expanded polyQ domain is a nuclear export signal, and further provided mechanistic explanation of how protein nuclear transport receptors modulate polyQ toxicity. / Chan, Wing Man. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0113. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 189-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Abstract not available

Bibliography: leaves 103-116. / vi, 116 leaves, [6] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Addresses the practical aspects of using nuclear transfer for the production of identical animals. Results from experiments provide improved understanding of the technical constraints of nuclear transfer. Also the flexibility of the methodology was increased with the use of in vitro culture and/or in vitro matured oocytes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1992

Four experiments were conducted to investigate methods of producing transgenic bovine embryos entirely in vitro. Experiment 1 examined the effect of DNA microinjection at 11, 15 and 19 h after fertilization (haf) on survival rate and DNA detection frequency by polymerase chain reaction (PCR). There was no difference in transgene detection frequency between treatments (53% at 11; 50% at 15; 48% at 19 haf). Of all injected embryos developing to the morula or blastocyst stage after 7 d in culture, 89% tested positive for the presence of the transgene by PCR. Greater developmental efficiencies can be obtained when injection is performed early in pronuclear formation (7% (11/161) at 11; 4% (61159) at 15; 1 % (1/165) at 19 haf; p<0.05). Experiment 2 examined the effect of microinjection of DNA into the germinal vesicle (gv) of bovine oocytes on subsequent development and detection of the transgene. Injection of the transgene into the gv reduced developmental rates compared to controls (control=23% (89/384); non-injected=9% (23/250); GV injected=5% (12/259); p<O.05). Transgene detection frequency was 64% (37/58). Injection of bovine oocytes before fertilization results in viable embryos containing the transgene, although at low frequencies. Experiment 3 was designed to examine whether the frequency of microinjected DNA detection by peR In whole bovine embryos would decline over a 21 d culture period. At d 0, the transgene was detected in 100% (46/46) of embryos analyzed. At d 7, detection frequency was 84% (51/62) in viable embryos, at d 14 49% (18/37), and at d 21 38% (3/8). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates until after 14 d in culture. Experiment 4 examined microinjected bovine embryos for their potential use as donor embryos in nuclear transfer, or cloning. There was no difference in development between embryos cloned from microinjected donor embryos and those from control donor embryos (injected=11 % (37/377); control=9% (7/81); p>0.05). Of the embryos developing from microinjected donors, 32% (12/37) were PCR positive. Microinjected embryos can be successfully used in a nuclear transfer program to produce more viable embryos, and the resulting embryos may be more reliably screened by PCR. The efficiency of producing viable bovine embryos positive for the injected gene may be increased by performing microinjection early in pronuclear formation, and entering the resulting embryos into a nuclear transfer program. / Ph. D.