Examination of a modified cell cycle synchronization method and bovine nuclear transfer using synchronized early G1 phase fibroblast cells
Abstract
Somatic cell nuclear transfer has a low success rate, due to a high incidence of fetal loss and increased perinatal morbidity/mortality. One factor that may affect the successful development of nuclear transfer embryos is the cell cycle stage of the donor cell. In order to establish a cell cycle synchronization method that can consistently produce cloned embryos and offspring, we examined the effects of different combinations of three cell treatments on the recovery rate of mitotic phase cells using bovine fetal fibroblasts. In the first experiment, we examined the recovery rate of mitotic phase cells by a combination of treatment with a metaphase arrestant (1 microM 2-methoxyestradiol), shaking the plate and selecting cells with a diameter of 20 microns. As a result, 99% of mitotic phase cells were recovered by repeating the combined treatment of metaphase arrestant and shaking, and collection of cells with a specific diameter. In the second experiment, nuclear transfer was carried out using early G1 phase cells by choosing pairs of bridged cells derived from mitotic phase cells recovered by the combined treatment of 1 microM 2-methoxyestradiol and shaking, and collection of cells with a diameter of 20 microns. The reconstruct...Continue Reading
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