| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2011: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Research Abstract |
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
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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
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