Modification of SCNT donor cell metabolism to mimic blastomere metabolism

描述（由申请人提供）：体细胞核移植（SCNT）是一个效率低下的过程。我们的目标是通过在SCNT之前和之后驱动成纤维细胞代谢向卵裂球代谢来改善SCNT胚胎的发育，作为改善克隆胚胎发育的一种方式。我们的首要论点是：SCNT之前的成纤维细胞或SCNT之后的胚胎，被驱动朝向卵裂球代谢，将使得从成纤维细胞到卵裂球代谢的转变不那么显著，并导致胚胎发育的改善。我们理想的方法是在基因修饰后或SCNT前一天开始处理细胞以诱导“卵裂球样”代谢。如果在选择遗传修饰期间有必要开始处理，那么我们必须确认它们被扩增、形成集落和在冷冻保存中存活的能力。具体目标是：1）将供体细胞的处理表征为更“卵裂球样”，2）在SCNT之前处理供体细胞，3）仅处理SCNT胚胎，4）处理供体细胞和/或SCNT胚胎，并评价足月发育。假设1：成纤维细胞可以被驱动向“卵裂球样”（WE）代谢。成纤维细胞将用几种化合物/条件单独和组合处理，这将驱使它们朝向卵裂球或瓦尔堡途径。假设二：具有“分裂球”代谢的成纤维细胞将产生比对照成纤维细胞衍生的胚胎具有更多“胚胎”代谢的SCNT胚胎，并且将具有更好的附着后发育。来自假设1的导致最“卵裂球样”代谢的处理将用于处理供体细胞以产生SCNT胚胎。假设3：被诱导具有“卵裂球样”代谢的SCNT胚胎将比对照胚胎发育得更好。这些实验将确定SCNT胚胎的处理是否会诱导更“卵裂球样”的代谢，并以更高的速度发育到附着后阶段。假设4：成纤维细胞和SCNT胚胎被驱动到“卵裂球样”代谢将导致SCNT胚胎比对照具有更强的发育能力。这项实验将联合收割机的努力，处理供体细胞变得更“卵裂球样”的处理，以加强发展的SCNT处理的胚胎。在本实验中，供体细胞将被处理或用作对照（基于假设2结果）。此外，SCNT胚胎将被处理或用作对照（基于假设3结果）。因此，我们提出了一个2X2阶乘处理结构。将胚胎培养至囊胚期并进行评价或转移。终点与假设3相同。总的来说，我们预计在SCNT之前调节供体细胞和/或在SCNT之后调节胚胎的代谢将导致胚泡阶段和发育的第35天之间的发育改善。

DESCRIPTION (provided by applicant): Somatic cell nuclear transfer (SCNT) is an inefficient process. Our goal is to improve the development of SCNT embryos by driving fibroblast metabolism toward blastomere metabolism before and after SCNT as a way to improve development of the cloned embryo. Our overarching thesis is: Fibroblasts prior to SCNT or embryos after SCNT, driven pharmacologically toward a blastomere metabolism will make the transition from fibroblast to blastomere metabolism less dramatic and result in improved development of the embryo. Our ideal methodology would be to treat the cells to induce a "blastomere-like" metabolism beginning after genetic modification, or alternatively the day before SCNT. If it is necessary to begin the treatment during selection for the genetic modification, then we must confirm their ability to be expanded, form colonies and survive cryopreservation. The Specific Aims will be to: 1) Characterize treatment of the donor cells to be more "blastomere- like", 2) Treat the donor cells prior to SCNT, 3) Treat the SCNT embryos only, 4) Treat the donor cells and/or the SCNT embryos, and evaluate development to term. Hypothesis 1: Fibroblast cells can be driven toward a "blastomere-like" (WE) metabolism. Fibroblast cells will be treated with several compounds/conditions, individually and in combination, that would drive them toward a blastomere or Warburg pathway. Hypothesis 2: Fibroblasts with a "blastomere" metabolism will result in SCNT embryos that have more of an "embryo" metabolism than those derived from control fibroblasts, and will have better post-attachment development. The treatment from Hypothesis 1 that results in the most "blastomere-like" metabolism will be used to treat donor cells to create SCNT embryos. Hypothesis 3: SCNT embryos induced to have a "blastomere-like" metabolism will develop better than control embryos. These experiments will determine if treatment of the SCNT embryo will induce a more "blastomere-like" metabolism and develop at a higher rate to post-attachment stages. Hypothesis 4: Fibroblasts and SCNT embryos driven to a "blastomere-like" metabolism will result in SCNT embryos that have more developmental competence than controls. This experiment will combine the efforts of treating the donor cells to become more "blastomere-like" with the treatment that enhances development of SCNT-treated embryos. In this experiment donor cells will be treated or used as controls (based on Hypothesis 2 results). In addition, SCNT embryos will be treated or used as controls (based on Hypothesis 3 results). Thus we propose a 2X2 factorial treatment structure. Embryos will be cultured to the blastocyst stage and evaluated or transferred. Endpoints will be identical to Hypothesis 3. Overall we expect that adjusting the metabolism of the donor cell prior to SCNT and/or the embryo after SCNT will result in improved development between the blastocyst stage and day 35 of development.