Function of centrosome at the first cleavage after nuclear transplantation

核移植是从体细胞创造克隆动物的关键技术。我们报道了在青竹鱼类克隆领域的第一次成功，使用了一种新的方法，即用来自成体尾鳍组织的原代培养细胞的供体核进行核移植。在这种方法中，供体核被转移到未去核和二倍体的受精卵中。随后重组胚胎的发育产生了与供体表型相同的后代，并且在体细胞胚系细胞的组成上不嵌合。然而，目前仍不清楚受体核去了哪里，以及为什么供体核选择保留在重建的胚胎中。为了回答这些问题，我们决定进一步调查，特别是来自受者和供者来源的中心体的不同贡献。在这个项目开始的时候，我们测试了核移植是否是供体核来源的真正克隆。为此，我们进行了…More利用遗传多样性的青山品系(d-RR)和供体(HNI-I)的组合进行了核移植，并成功地获得了三例成人核移植。这两个菌株的DNA多态性使我们能够确定DNA的来源。采用聚合酶链式反应(PCR)多态标记技术检测核移植的基因分型。在所有染色体位置上总共检查了96个标记，24条染色体中每条染色体上都有4个标记。所有来自三个移植物的鳍和一个移植的内脏的基因组DNA样本都显示出与HNI-I株相同的基因。这些结果表明，这三个移植的细胞核来自供者。接下来，我们试图研究来自受体和供体来源的中心体的不同贡献。然而，克隆效率(In？)青竹的价格极低。我们设计了一种不同的方法来提高核移植的效率，使用多能细胞作为供体。获得了河豚Oct3/4启动子驱动的转基因青蛙TG(Oct3/4：EGFP)。Oct3/4(Pou5f1)在哺乳动物的多能干细胞中特异表达，对其自我更新和维持是不可或缺的。GFP的表达模仿了内源Oct3的表达，因此Fugu Oct3调控序列的2kb片段足以再现青冈Oct3的内源表达模式。转基因亲本的后代在胚胎发育早期的所有卵裂球和胚泡中都呈GFP阳性，直到体节后期，胚体的大部分部位都是荧光的。较少

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