Understanding mechanism and improving efficiency of somatic cell nuclear transfer

了解体细胞核移植的机理和提高其效率 摘要 了解细胞命运重编程的机制对于基础生物学和再生生物学都很重要。 药在目前可用的重编程技术中，体细胞核移植（SCNT）是唯一的 一种允许将终末分化细胞有效和快速重编程为全能合子样的方法， 状态全能性是细胞产生生物体及其胎盘组织的能力。但尽管 自SCNT第一次成功克隆以来，经过50多年的努力， 了解SCNT重编程是如何实现的。虽然某些多能性的异位表达 转录因子（TF）可以将体细胞重编程为诱导多能干细胞（iPSC），这些细胞 不是全能的。此外，越来越多的证据表明，SCNT介导的重编程， 这与基于转录因子的iPSC重编程的机制不同。自从维护 处于谱系不受限制的幼稚状态的未分化干细胞对于治疗目的是重要的， 了解分化的体细胞如何重新编程为全能状态既具有生物学意义， 临床重要性 在SCNT介导的重编程过程中，供体细胞基因组关闭其细胞类型特异性转录 程序，并采用一种新的基因表达谱，模仿全能合子。我们的初步研究 表明供体细胞的转录重编程在SCNT后12小时内完成， 这表明卵母细胞中存在的母体因子可以在细胞分裂后迅速重置体细胞的染色质状态， 核移植基于这一有趣的观察，以及我们最近开发的技术， 分析小鼠受精卵的染色质可及性并进行母体因子去除，我们建议 了解SCNT重编程的机制，并通过以下具体措施提高SCNT效率 目的： 1)鉴定和测试SCNT重编程所需的TF和染色质重塑因子; 2)克服SCNT胚胎发育缺陷，提高动物足月率。 完成拟议的研究不仅将确定卵母细胞因子对SCNT介导的 重编程，而且还提高了SCNT效率，以实现最大的长期率。这些成果将 在发育、干细胞、生殖细胞、染色质生物学等领域具有深远的影响， 再生医学

Understanding mechanism and improving efficiency of somatic cell nuclear transfer (SCNT) Abstract Understanding the mechanism of cell fate reprogramming is important for both basic biology and regenerative medicine. Of the currently available reprogramming techniques, somatic cell nuclear transfer (SCNT) is the only one that allows efficient and rapid reprogramming of terminally differentiated cells to the totipotent zygote-like state. Totipotency is the ability of a cell to give rise to an organism and its placental tissues. However, despite more than 50 years of effort since the first successful cloning by SCNT, very little progress has been made in understanding how SCNT reprogramming is achieved. Although ectopic expression of certain pluripotency transcription factors (TFs) can reprogram somatic cells into induced pluripotent stem cells (iPSCs), these cells are not totipotent. Moreover, accumulating evidence suggest that SCNT-mediated reprogramming is mechanistically different from that of transcription factor-based iPSC reprograming. Since maintaining undifferentiated stem cells in a lineage-unrestricted naïve state is important for therapeutic purposes, understanding how differentiated somatic cells are reprogrammed into a totipotent state is of both biological and clinical importance. During SCNT-mediated reprogramming, donor cell genomes turn off their cell-type specific transcription programs and adopt a new gene expression profile that mimics that of totipotent zygotes. Our preliminary studies indicate that transcriptional reprogramming of donor cells is accomplished within 12 hours following SCNT, indicating that maternal factors present in oocytes can reset the chromatin state of somatic cells quickly upon nuclear transfer. Building upon this intriguing observation, as well as our recently developed techniques in analyzing chromatin accessibility of mouse zygotes and performing maternal factor depletion, we propose to understand the mechanism of SCNT reprogramming and improve SCNT efficiency with the following specific Aims: 1) Identifying and testing TFs and chromatin remodeling factors required for SCNT reprogramming; 2) Overcoming SCNT embryo developmental defects to increase animal term rate. Completion of the proposed study will not only identify oocyte factors important for SCNT-mediated reprogramming, but also improve the SCNT efficiency to achieve maximum term rate. These achievements will have far-reaching implications in the fields of development, stem cell, germ cell, chromatin biology, and regenerative medicine.