Mouse somatic cell clones: Reprogramming and development

小鼠体细胞克隆：重编程和发育

• 批准号: 6839435
• 负责人: Kenneth J McLaughlin
• 金额: $ 28.53万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6839435
• 关键词: antineoplastics; chromosomes; clone cells; egg /ovum; embryo /fetus; growth /development; laboratory mouse; microtubules; mitotic spindle apparatus; molecular assembly /self assembly; molecular cloning; polymerization; tissue mosaicism

DESCRIPTION (provided by applicant): Factors relevant and possibly limiting to the development of somatic cell clones include procedure, type of donor nucleus, and recipient oocyte. Preparation of the ooplast for nuclear transfer involves a procedure that removes chromosomes and, inadvertently, the meiotic spindle and surrounding cytoplasm. This results in elimination of the oocyte genome, but is presumably associated with the loss of factors beneficial for the development of the clone after introduction of the somatic cell nucleus. Preliminary findings indicate that oocyte chromosome removal after brief treatment with microtubule depolymerizing drugs improves mouse clone development. We will therefore examine in detail, the disassembly and assembly of the spindle under the influence of microtubule inhibitors in both murine and bovine oocytes to determine the kinetics of spindle disassembly during chromosome removal. Optimal parameters for spindle retention during chromosome removal will be assessed for developmental consequences. Identifying the timing of such events will provide the basis for future studies to identify what factors associated with the spindle are developmentally beneficial for somatic cell clones. Aside from the recipient ooplasm, properties of the somatic donor nucleus influence development apparent in the development and phenotypes of clones from different cell types. It unknown if differences exist between donor nuclei of the same population, allowing the development of a small number of clones. This should be reflected in the developmental diversity of clones derived from the same cell type. However, the early developmental demise of the majority of somatic cell clones precludes a full examination. Our preliminary data indicate that development of clones can be extended into later development by aggregation with other clones or normal embryos. The low total cell number of blastocyst stage clones also suggests that clone failure may be related to a proliferation defect early in development. In Specific Aim 2 we will determine the extent of developmental contribution potential by producing chimeras of clones with normal embryos. The number clones that can contribute to preimplantation and fetal development will be ascertained, as well as the extent and tissue-type of contribution. Both assessment of clone contribution in chimeras and the relevance of spindle-associated factors will provide novel basic information about reprogramming of somatic cell nuclei.

描述（由申请人提供）：与体细胞克隆发展相关和可能限制的因素包括程序，供体核类型和受体卵母细胞。卵母体的核转移准备过程包括染色体和减数分裂纺锤体及其周围的细胞质的去除。这导致卵母细胞基因组的消除，但可能与引入体细胞细胞核后有利于克隆发育的因素的丧失有关。初步结果表明，微管解聚药物短暂治疗后卵母细胞染色体去除可改善小鼠克隆发育。因此，我们将在小鼠和牛卵母细胞中详细检查纺锤体在微管抑制剂的影响下的拆卸和组装，以确定染色体去除过程中纺锤体拆卸的动力学。染色体去除过程中纺锤体保留的最佳参数将评估发育后果。确定这些事件的时间将为未来的研究提供基础，以确定与纺锤体相关的哪些因素对体细胞克隆的发育有益。除了受体卵浆外，体细胞供体核的特性还会影响不同细胞类型克隆的发育和表型。目前尚不清楚同一种群的供体细胞核之间是否存在差异，从而允许少量克隆的发展。这应该反映在来自同一细胞类型的克隆的发育多样性上。然而，大多数体细胞克隆的早期发育死亡阻碍了全面的检查。我们的初步数据表明，克隆的发育可以通过与其他克隆或正常胚胎聚集而延长到后期发育。囊胚期克隆的低细胞总数也提示克隆失败可能与发育早期的增殖缺陷有关。在特异性目标2中，我们将通过产生具有正常胚胎的克隆嵌合体来确定发育贡献潜力的程度。将确定能够促进着床前和胎儿发育的克隆数量，以及贡献的程度和组织类型。对嵌合体克隆贡献的评估和纺锤体相关因子的相关性将为体细胞细胞核重编程提供新的基础信息。