한빛사 논문
Abstract
Eunju Kang1, GuangmingWu2, Hong Ma1, Ying Li1, Rebecca Tippner-Hedges1, Masahito Tachibana1†, Michelle Sparman1, Don P. Wolf1, Hans R. Schöler2 & Shoukhrat Mitalipov1
1Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon 97006, USA. 2Max Planck Institute for Molecular Biomedicine, Mu¨ nster 48149, Germany. †Present address: South Miyagi Medical Center, Miyagi 989-1253, Japan.
Correspondence to: Shoukhrat Mitalipov
Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei1, 2, 3. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive4, 5. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT6, 7, 8, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are ‘trapped’ inside the nucleus during interphase and effectively removed during enucleation9. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.
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