Mouse Somatic Nuclear Transfer Without Micromanipulators

Mouse somatic cell nuclear transfer was an important method for foundational research in development and genetics, and was very important for our understanding the mechanism of clone also. These studies focus on developing a mouse somatic cell nuclear transfer procedure without micromanipulators, and perfecting the common mouse somatic cell nuclear transfer procedure to reduce techniques required and increases the efficiency. This somatic cell nuclear transfer procedure without micromanipulators involved in somatic cell culture, activation of reconstructed embryos, embryos in vitro culture and embryos transplantation, and all these may have some similarity or specialty compare with common somatic cell nuclear transfer procedure, such as the in vitro culture of zona-free embryos and embryos transplantation. The main results were followed: 1. Oocytes enucleation was one of the key factors to decides the successes of animal clone. We have studied several factors (including ethanol-activated oocytes treated with demecolcine at different times post-activation, concentration of demecolcine, culture time after activation and age of oocytes) that effected demecolcine-induced enucleation. The results indicated: 1) While the 0.4 μg/ml demecolcine was used to induce enucleation, the highest induce-enucleated rate (29.7%) was acquired while ethanol-activated oocytes treated with demecolcine at immediately post-activation. 2) Prolong culture time can not increase demecolcine induce-enucleated rate, the effectors of demecolcine are reversible. 3) Concentration of demecolcine effect induce-enucleated rate, the highest induce-enucleated rate (33.4%) was acquired while ethanol-activated oocytes treated with demecolcine for 45 min at immediately post-activation. 4) Oocytes collected after hCG injected 18 h were more suitable for demecolcine induce-enucleated. In conclusion, the best conditions of Kun-ming strain mouse oocytes demecolcine induce-enucleated was: oocytes collected after hCG injected 18 h were treated with 0.6 μg/ml demecolcine for 45 min at immediately post-activated with 7% (v/v) ethanol. 2. One of the important factors that determine the success of the development of cloned embryos is the cell cycle stage of the cloning donor cells. We culture and passages mouse ear fibroblast, and examine the effect of different treatment on cell cycle. On the same time, to preserve the somatic cell germplasm use for nuclear transfer, we developed a micro-cryopreservation method. The results indicated: The cell cycle distribution and percentage of cell apoptosis was not different among 1-7 passages cell. The confluence and touch inhibition all affect the cell cycle distribution and percentage of cell apoptosis. Cell in 70-80% confluence was more suitable use serum starve to gather cells into G0/G1 phase. Treated cells with 13.5μM demecolcine for 14h can gather culture cells into G2/M phase efficiently. While storage time no more than 180 days, living cells percentage of micro-cryopreserved cell was not affected. The efficiency of micro-cryopreservation was different for cells in different cell cycle, after storage the living cells percentage of G0/G1 phase cells was higher than G2/M phase cells. Both passages (1-9 passages) and micro-cryopreservation have none effects on cell chromosome number. 3. Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10 mM SrCl2 for 30 min-5 h, activation rates (88.4±4.1 to 91.2±2.7%) did not differ (P>0.05), but rate of blastulation (57.3±3.5%) and cell number per blastocyst (45.0±2.4) were the highest after 2.5 h treatment. When they were treated with 1-20 mM SrCl2 for 2.5 h, the activation rate and cell number per blastocyst were higher (P<0.05) after 10 mM SrCl2 treatment than other treatments. The best activation and development was obtained with Ca2+-free Sr2+ medium but the activation rate was low (37.7±1.6%) in Ca2+-containing medium. Activation rates were the same regardless of the presence or absence of cytochalasin B (CB) in activating medium but the blastulation rate was higher (P<0.05) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6 mM SrCl2, and many lysed with increased intensity of Sr2+ treatment. The presence of CB in SrCl2 medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr2+ treatment alone, with 34% blastulating, but rates of activation and blastulation increased (P<0.05) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less (P<0.05) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl2 treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain thebest activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5 h with 10 mM SrCl2 in Ca2+-free medium supplemented with 5μg/mL CB. 4. The culture of zona-free embryos was the issue must have been solved before carry thorough the zona-free oocytes nuclear transfer. We determines the zona-free primarily nuclear stage embryos development culture in different culture system, seven culture system were used, including multiplex oocytes culture, micro-drops single oocyte culture, micro-drops single oocyte add multiplex oocytes culture, glass capillary culture, the well of the well (WOW) culture, agarose-holes embed culture, and agarose-pillar embed culture. The results indicated, the agarose-pillar embed culture was the best system for zona-free embryos (61% embryos developed into blastocyst, 92% zona-free blastocyst can be recovered, the average cell number of each blastocyst was 45); in despite of the blastocyst development rate was low than zona-intact embryos culture in multiplex oocytes culture system still. 5. To developing a zona-free oocytes somatic cell nuclear transfer procedure to suit mouse, we studies several related issues with Kun-ming strain mouse. The results indicated: 1) Digest after serum-covered can remove zona pellucida of oocytes efficiently without increase lysis of oocytes. 2) The best condition of oocytes bisection was to use HEPES-CZB supplemented 2.5 μg/ml CB as manipulate medium and demi-oocytes storege at room remperature (about 25 oC). 3) The best fusion rate of dimeoocyte-somatic cell complexes was acquired with single direct current (DC) pulse of 60v/mm and 10μs duration, while the best fusion rate of dimeoocyte and dimeoocyte-somatic cell fused complexes was acquired with single direct current (DC) pulse of 45v/mm and 10μs duration. 4) After activation, 80% formed pseudo primarily nuclear. 51% reconstructed embryos developed into 2-cell in the agarose-pillar embed culture system, 9% developed into 4-cell, and none reconstructed embryos develop exceeded 4-cell. In conclusions, we primary developed a mouse zona-free oocytes somatic cell nuclear transfer techniques system. 6. To determine the effects of oocytes strain and donor somatic cell type on reconstructed embryos development, we directly injected C57BL/6 female mouse ear fibroblast nuclear into different strains (Kun-ming, C57BL/6♀×Kun-ming♂, 129/SvJ, C57BL/6♀×129/SvJ♂and B6D2F1) mouse enucleated MII-stage oocytes under the Pizol assistant, we also compared the development of reconstructed embryos which injected fibroblast or cumulus cell into B6D2F1 mouse oocytes. The results indicated: The nuclear transfer success rate...