Kurome Mayuko, Fujimura Tatsuya, Murakami Hiroshi, Takahagi Yoichi, Wako Naohiro, Ochiai Takashi, Miyazaki Koji, Nagashima Hiroshi
Laboratory of Developmental Engineering, Department of Life Science, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan.
Cloning Stem Cells. 2003;5(4):367-78. doi: 10.1089/153623003772032862.
This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.
本文在方法学上比较了电融合（EF）和胞质内注射（ICI）方法，以及同时融合/激活（SA）和延迟激活（DA），用于猪胎儿成纤维细胞的体细胞核移植。通过ICI或EF进行核移植后，对供体细胞核重塑模式的比较表明，无论融合培养基中是否存在Ca2+，两种情况下均有较高比例（80-100%）的染色体过早凝聚发生。ICI方法进行核移植时假原核的形成倾向较低（65%对85-97%，p<0.05）。使用EF方法用体外成熟（IVM）卵母细胞重构的核移植胚胎的体外发育潜力高于ICI方法产生的胚胎（囊胚形成率：19%对5%，p<0.05），并且使用体内成熟卵母细胞作为受体细胞质时并未得到改善。使用SA方案产生的胚胎发育到囊胚的效率与DA方案产生的胚胎相同（11%对12%）。基于产生克隆正常猪胎儿，EF方法与SA结合使用被证明是一种生产克隆猪的有效方法。然而，SA和DA方案之间核重塑模式的细微差异可能意味着它们的核重编程效率存在差异。
