Function of centrosome at the first cleavage after nuclear transplantation

核移植是利用体细胞克隆动物的关键技术。我们报道了一种新的方法，利用来自成年尾鳍组织的原代培养细胞的细胞核进行核移植，成功地克隆了米达卡鱼。在这种方法中，将供体细胞核移植到未去核和二倍体的受体卵子中。重建胚胎的后续发育产生了与供体表型相同的后代，并且在体细胞系细胞的组成上不嵌合。然而，目前尚不清楚受体细胞核去了哪里，以及为什么供体细胞核选择保留在重建的胚胎中。为了回答这些问题，我们决定进一步研究，特别是来自受体和供体来源的中心体的差异贡献。在这个项目开始的时候，我们测试了核移植是否是真正的供体核克隆。为此，我们利用遗传多样性medaka菌株（d-rR）和供体（HNI-I）的组合进行了更多的核移植，成功获得了3例成人核移植。这两个菌株的DNA多态性使我们能够确定DNA的起源。采用PCR多态标记检测移植物的基因型。在所有染色体位置共检测了96个标记，24条染色体各4个。3例移植鱼鳍和1例移植内脏的基因组DNA样本均与HNI-I株基因型一致。结果表明，3例移植细胞核均来源于供体。接下来，我们试图研究来自受体和供体来源的中心体的差异贡献。然而，medaka的克隆效率极低。我们设计了一种不同的方法，以多能性细胞作为供体来提高核移植的效率。获得了河豚oct3/4启动子驱动的转基因medaka Tg（oct3/4:egfp）。Oct3/4 （Pou5f1）在多能干细胞中特异性表达，是哺乳动物多能干细胞自我更新和维持所必需的。gfp表达模拟了内源性oct3的表达，因此河豚oct3调控序列的2kb片段足以再现medaka oct3的内源性表达模式。转基因亲本子代在胚胎发生早期所有卵裂球和胚皮均呈GFP阳性，胚体的大部分部位直到somte后期都呈荧光。少

Nuclear transfer is a key technique to create a cloned animal from a somatic cell. We reported the first success in the fish cloning field in medaka, using a novel method of nuclear transfer with donor nuclei of primary culture cells from adult tail fin tissues. In this method, the donor nuclei were transferred into non -enucleated and diplodized recipient eggs. Subsequent development of reconstructed embryo gave rise to offspring phenotypically identical to the donor, and not chimeric in the composition of somatic orgerm line cells. However, it is still unknown where the recipient nucleus has gone, and why the donor nucleus has chosen to be retained in the reconstructed embryos. In order to answer these questions, we decided to investigate further, especially the differential contribution of centrosomes from the recipient and donor sources. At the beginning of this project, we tested if the nuclear transplants were authentic clones of donor-nucleus origin. For this purpose, we conduct … More ed the nuclear transfer using a combination of genetically-diverse medaka strain (d-rR) and donor (HNI-I), and successfully obtained three adult nuclear transplants. The DNA polymorphisms of these two strains enabled us to identify the origin of DNA. We checked the genotype of the nuclear transplants by PCR polymorphic markers. A total of 96 markers were examined at all chromosomal locations, four on each of the 24 chromosomes. All the genomic DNA samples from the fin of the three transplants and from the internal organs of one transplant showed an identical genotype to HNI-I strain. These results indicated that the nuclei of the three transplants originated from the donor.Next, we tried to investigate the differential contribution of centrosomes from the recipient and donor sources. However, cloning efficiency of(in?) medaka is extremely low. We designed a different approach to improve the efficiency of nuclear transfer by using pluripotent cells as donor. The fugu oct3/4 promoter-driven transgenic medaka Tg(oct3/4:egfp) was generated. Oct3/4 (Pou5f1) is expressed specifically in pluripotent stem cells, and is indispensable for their self-renewal and maintenance in mammals. GFPexpression mimicked the endogenous oct3 expression, and thus the 2kb fragment of Fugu oct3 regulatory sequence is sufficient for reproducing the endogenous expression pattern of medaka oct3. The offspring of the transgenic parents were GFP -positive in all blastomeres and blastoderms during early embryogenesis, and most parts of the embryonic body were fluorescent until late somite-stage. Less