Therapeutic Cloning in Parkinsonian Mice

描述(由申请人提供)： 该项目的总体目标是证明治疗性克隆在帕金森病小鼠身上的可行性。干细胞和克隆技术受到了公众和科学的极大关注。干细胞的前景是为再生医学产生无限数量的特定细胞。克隆可以产生基因完全相同的生物体。治疗性克隆结合了这些承诺，为移植治疗产生了无限来源的自体专门细胞。然而，在实现这一目标的道路上仍然存在许多障碍，如核移植胚胎干细胞的高效生成、核移植胚胎干细胞向具有治疗潜力的特化细胞的终末分化以及对核移植胚胎干细胞后代的体内安全性和功能的评估。我们的提案将解决这些关键问题中的几个。 我们最近已经成功地从成年小鼠身上产生了NTES细胞，并在体外将这种细胞诱导成了中脑多巴胺神经元(Science 2001,292：740-3)。在这里，我们建议从成年帕金森病小鼠的尾尖活检组织中获得自体NTES细胞。在这些研究中，将使用转基因Z/EG双报告小鼠。这些小鼠结构性地表达LacZ，但在Cre介导的重组后转换为EGFP表达，从而在自体移植的背景下允许移植物和供体之间的遗传差异。在衍生后，MES系将受到带有PMC-Cre表达构建体的电穿孔。成功表达EGFP的克隆将被选择，在体外增殖，并使用先前定义的5步体外方案转化为多巴胺神经元。神经特异性表面分子PSA-NCAM的荧光激活细胞分选(FACS)将用于消除任何剩余的未分化细胞群。非侵入性的多巴胺测量将允许在植入前对NTES后代进行功能筛选。产生足够水平的多巴胺的细胞将被移植回为NTE生成(自体移植)提供成年细胞的同一小鼠的纹状体。行为学和组织学参数将用于评估治疗功能以及体内存活的NTES后代的数量和类型。这项研究是在小鼠身上进行治疗性克隆的首次尝试，并可能为这种方法在其他疾病中的应用铺平道路。该提案是按照R21应用的精神设计的，使用创新的策略来证明新的高影响技术在帕金森病实验性治疗中的可行性

DESCRIPTION (provided by applicant): The overall goal of this project is to demonstrate the feasibility of therapeutic cloning in Parkinsonian mice. Stem cell and cloning technologies have received significant public and scientific attention. The promise of stem cells is the generation of unlimited numbers of specific cells for regenerative medicine. Cloning allows the generation of genetically identical organisms. Therapeutic cloning combines these promises towards generating an unlimited source of autologous, specialized cells for transplantation therapy. However, substantial hurdles remain on the path towards that end such as efficient generation of nuclear transfer embryonic stem (ntES) cells, terminal differentiation of ntES cells into specialized cells of therapeutic potential and assessment of in vivo safety and function of ntES cell progeny. Our proposal will address several of these key issues. We have recently succeeded in the generation of ntES cells from adult mice and in coaxing such cells into midbrain dopamine neurons in vitro (Science 2001,292:740-3). Here we propose to derive autoiogous ntES cells from tail tip biopsies of adult Parkinsonian mice. For these studies transgenic Z/EG double reporter mice will be used. These mice constitutively express lacZ but switch to eGFP expression upon Cre-mediated recombination, thus allowing genetic distinction between graft and donor within the context of an autologous graft. Upon derivation MES lines will be subjected to electroporation with the pMC-Cre expression construct. Successful eGFP expressing clones will be selected, proliferated in vitro and converted into dopamine neurons using a previously defined 5-step in vitro protocol. Fluorescence activated cell sorting (FACS) for the neural-specific surface molecule PSA-NCAM will be used to eliminate-any remaining undifferentiated cell populations. Non-invasive dopamine measurements will allow functional screening of ntES progeny prior to implantation. Cells producing adequate levels of dopamine will be transplanted back into the striaturn of the same mice that provided adult cells for ntES generation (autologous grafts). Behavioral and histological parameters will be used to assess the therapeutic function as well as the number and types of surviving ntES progeny in vivo. This study is the first attempt of therapeutic cloning in mice and might pave the way for the application of this approach in other diseases. The proposal is designed in the spirit of the R21 application using innovative strategies to demonstrate the feasibility of novel high-impact techniques in the experimental treatment of Parkinson's disease