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The Isolation And Characterization Of Bovine Adult Derived Adipose Stem Cells For The Use In Nuclear Transfer, Alicia A. Picou
The Isolation And Characterization Of Bovine Adult Derived Adipose Stem Cells For The Use In Nuclear Transfer, Alicia A. Picou
LSU Master's Theses
Since the cloning of Dolly there has been little change in the efficiency of nuclear transfer (NT). Research is beginning to investigate the characteristics of donor cells. Adiposetissue is an abundant source of adult-derived cells that have displayed “stemness” in-vitro(Gimble et al., 2003). The overall goal of this research was to define the in-vitro characteristicsof bovine adipose-derived adult stem cells (ADAS) for the use in NT. Isolation methods weredetermined by a 3 x 3 factorial design. 1 g of subcutaneous fat was collected and subjected to0.10%, 0.25% or 0.50% collagenase type I solution for 1, 2 and 3 h. Nucleated …
Chromosomal Stability And Epigenetic Modifications Of Fibroblast Cells Used For Nuclear Transfer, Angelica Maria Giraldo Gomez
Chromosomal Stability And Epigenetic Modifications Of Fibroblast Cells Used For Nuclear Transfer, Angelica Maria Giraldo Gomez
LSU Doctoral Dissertations
Investigations into the importance of the nuclear integrity of the donor cell prior to nuclear transfer (NT) are limited. In Experiment 1, the proliferative characteristics, chromosomal stability and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation at different population doublings (PDs) were investigated. The cells divided at a constant rate and cell cycle length increased only at the end of the proliferative stage. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. High levels of multinucleated cells and abnormal spindle configurations were …
Somatic Cell Interspecies Nuclear Transfer, Marina Julia Sansinena
Somatic Cell Interspecies Nuclear Transfer, Marina Julia Sansinena
LSU Doctoral Dissertations
The low efficiency of the nuclear transfer (NT) procedure requires large number of oocytes to produce embryos and live offspring. A series of experiments were conducted to evaluate the ability of the bovine cytoplast to reprogram nuclei from horses and llamas. In a preliminary study, equine oocytes from small (<20mm diameter) follicles were either pretreated with roscovitine or placed in maturation (IVM only) prior to NT. Roscovitine pretreatment did not improve nuclear maturation rates (roscovitine pretreatment 57% vs. IVM only 66%) and no fusion was obtained from roscovitine-pretreated oocytes after NT. Another preliminary study was conducted with the objective to produce llama NT embryos and to compare their development in two in vitro culture conditions (G1.2® vs. CR1aa). No difference was found in the number of embryos cleaved after 2 d of culture. This resulted in the first scientific report of somatic cell NT, in vitro culture and transfer of NT embryos in the llama. In the next experiment, adult horse and llama fibroblasts were injected into enucleated cow oocytes. The results showed the cow cytoplasm is capable of partially reprogramming nuclei from other species and support mitotic divisions. However, this study also showed a consistent embryonic developmental arrest at the 8- to 16- cell stage when horse or llama donor cells were used as donor nuclei. When a more closely related species of donor cell (banteng) and recipient oocyte (domestic cattle) were used for NT, no embryonic developmental arrest was found. Embryos progressed to achieve high blastocyst rates (banteng male cell line 28% vs. banteng female cell line 15%). Two banteng interspecies NT pregnancies were established and subsequently lost from the banteng male cell line. In the final study, the effect of a mixed mitochondrial population (heteroplasmy) on early embryonic development was investigated. Ooplasmic transfer performed in combination with NT procedure indicated presence of foreign mitochondria clustered in a small portion of the cytoplasm in early stages of embryo development. When goat ooplasm was transferred into interspecies (cow oocyte-goat donor cell) NT embryos, fusion and cleave rates were reduced suggesting an increased level of heteroplasmy or nuclear-ooplasmic incompatibilities.
Production Of Transgenic Goats By Somatic Cell Nuclear Transfer, Brett C. Reggio
Production Of Transgenic Goats By Somatic Cell Nuclear Transfer, Brett C. Reggio
LSU Doctoral Dissertations
A series of experiments were conducted to produce transgenic goats by somatic cell nuclear transfer (NT). In all experiments, donor cells were electrically fused to enucleated metaphase II oocytes, then chemically activated. In a preliminary study to evaluate embryonic development following NT with proliferating or quiescent fibroblast or cumulus cells, significantly more embryos reconstructed with quiescent cumulus cells fused (77%) compared with proliferating cumulus cells (41%), proliferating fibroblasts (36%) or quiescent fibroblasts (37%). Improved development to the eight- to sixteen-cell stage was observed when fibroblast cells were serum starved (serum starved 39% vs. serum fed 15%). However, there was no …