Understanding mechanism and improving efficiency of somatic cell nuclear transfer

体细胞核移植(SCNT)机制的理解和效率的提高 摘要 了解细胞命运重编程的机制对基础生物学和再生都很重要 医药。在目前可用的重编程技术中，体细胞核移植(SCNT)是唯一 一种能够高效、快速地将终末分化细胞重新编程为全能受精卵样细胞的方法 州政府。全能性是指细胞产生生物体及其胎盘组织的能力。然而，尽管 自SCNT第一次成功克隆以来，经过50多年的努力，在 了解如何实现SCNT重新编程。尽管某些多能性的异位表达 转录因子(TF)可以将体细胞重新编程为诱导的多能干细胞(IPSCs)，这些细胞 并不是全能的。此外，越来越多的证据表明，SCNT介导的重新编程是 在机制上不同于基于转录因子的IPSC重新编程。自维护以来 处于不受血统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被重新编程为全能状态既是生物学上的，也是 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了其细胞类型的特定转录 计划并采用一种新的基因表达谱，模仿全能受精卵的基因表达谱。我们的初步研究 表明供体细胞的转录重新编程在SCNT后12小时内完成， 这表明，卵母细胞中存在的母性因素可以迅速重置体细胞的染色质状态 核转移。基于这一有趣的观察结果，以及我们最近在 分析小鼠受精卵的染色质可及性，并进行母体因子耗竭，我们建议 了解SCNT重新编程的机制，并通过以下具体操作提高SCNT的效率 目标： 1)鉴定和检测SCNT重编程所需的转录因子和染色质重塑因子； 2)克服SCNT胚胎发育缺陷，提高动物足月率。 拟议研究的完成不仅将确定对SCNT介导的卵母细胞重要的因素 重新编程，还可以提高SCNT的效率，实现最大期限速率。这些成就将 在发育、干细胞、生殖细胞、染色质生物学和 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.