Chen C-H, Stone L, Ju J-C, Lien W-T, Liu M-S, Tu C-F, Lee K-H
Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan.
Reprod Domest Anim. 2008 Oct;43(5):547-55. doi: 10.1111/j.1439-0531.2007.00951.x. Epub 2008 Feb 25.
Most studies of mouse cloning successfully achieved activation of the reconstructed oocytes by strontium (Sr) combined with cytochalasin B (CB) treatment. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was used to inhibit the activity of maturation promoting factor for activation of oocytes, but it has never been successfully applied in mouse cloning. This study investigates the activation efficiency of 6-DMAP in mouse somatic cell nuclear transfer (SCNT). Higher parthenogenetic blastocyst rates (71-72%, p < 0.05) were achieved in the oocytes treated with Sr6D (10 mM Sr combined with 2 mM 6-DMAP for 4 h) and Sr6D + SrCB (Sr6D for 2 h then Sr combined with 5 mug/ml CB for another 2 h), and a higher rate of hatching and hatched blastocyst was observed in the Sr6D + SrCB group (31%, p < 0.01) compared with other treatment groups (1-8%). For mouse cloning, cumulus cells of enhanced green fluorescent protein (EGFP)-expressed ESC chimera F1 were used as donor nuclei. Following activation, better development of the cloned embryos was observed in Sr6D + SrCB treatment. Moreover, different media, i.e. KSOM-AA, MEM-alpha and MK, for culturing cloned embryos were also compared in this study. Better morula/blastocyst (40%) and blastocyst (29%) rates were achieved in the embryos cultured in MEM-alpha medium (p < 0.05). Consequently, four EGFP cloned mice were generated in the activation treatment containing 6-DMAP following embryo transfer. In conclusion, treatment with 6-DMAP in combination with other activation stimuli successfully activates mouse reconstructed oocytes and support full-term development of the transgenic SCNT cloned embryos.
大多数小鼠克隆研究通过用锶（Sr）联合细胞松弛素B（CB）处理成功实现了重构卵母细胞的激活。一种蛋白激酶抑制剂，6-二甲基氨基嘌呤（6-DMAP），曾被用于抑制成熟促进因子的活性以激活卵母细胞，但它从未在小鼠克隆中成功应用过。本研究调查了6-DMAP在小鼠体细胞核移植（SCNT）中的激活效率。在用Sr6D（10 mM Sr与2 mM 6-DMAP联合处理4小时）和Sr6D + SrCB（Sr6D处理2小时，然后Sr与5 μg/ml CB联合处理另外2小时）处理的卵母细胞中，孤雌胚泡率更高（71 - 72%，p < 0.05），并且与其他处理组（1 - 8%）相比，在Sr6D + SrCB组中观察到更高的孵化和孵化胚泡率（31%，p < 0.01）。对于小鼠克隆，表达增强绿色荧光蛋白（EGFP）的胚胎干细胞嵌合体F1的卵丘细胞被用作供体细胞核。激活后，在Sr6D + SrCB处理中观察到克隆胚胎有更好的发育。此外，本研究还比较了用于培养克隆胚胎的不同培养基，即KSOM-AA、MEM-α和MK。在MEM-α培养基中培养的胚胎获得了更好的桑椹胚/胚泡率（40%）和胚泡率（29%）（p < 0.05）。因此，在胚胎移植后，在含有6-DMAP的激活处理中产生了四只EGFP克隆小鼠。总之，6-DMAP与其他激活刺激联合处理成功激活了小鼠重构卵母细胞，并支持转基因SCNT克隆胚胎的足月发育。
