Biopsy and Freezing of Later-stage Mouse Blastocysts Using the Dracula Pipette

描述(申请人提供)：植入前胚胎的程序是许多生物医学研究的基石；然而，在所有情况下，程序都非常困难和精细，需要非常熟练的技术专家和昂贵的设备。与这些程序相关的困难往往导致大多数生物医学研究小组回避大多数此类工作。当必须执行时，通常将其外包给专门的实验室或设施。这样做的两个缺点是：(1)由于这些程序的成本和难度，许多重要的问题可能会被避免；(2)由于冷冻保存的成本和/或复杂性令人望而却步，许多独特的、具有潜在价值的小鼠品系可能会丢失或被放弃。GeneSearch公司已经开发出一种新的同轴胚胎操作工具，有望解决这些问题。这种工具的创新之处在于其简单的设计，德古拉不是在胚胎的相反两极使用单独的“握持”和“操纵”吸管，而是在握持吸管内有操纵吸管。主要的优点是，操作吸管接触胚胎的区域受到由保持吸管施加的调节力的限制。该工具的原始原型首次实现了戏剧性的进步 在大的孵化胚胎的超低温保存中，如骆驼和马的胚胎。现在，GeneSearch正在缩小工具的尺寸，在一些部件上使用新材料，并改进专门用于操纵老鼠大小的胚胎的操作。拟议研究的第一阶段旨在优化用于小鼠胚胎程序的“Mouse Dracula”工具，包括胚胎冷冻、通过ES细胞注射产生嵌合体小鼠，以及用于遗传分析的存活活组织检查。为此，我们提出了三个具体目标，即(1)优化工具性能，(2)开发改进的小鼠胚胎冷冻保存方案，(3)测试该工具用于嵌合体生产和基于存活活检的遗传分析的性能。这是蒙大拿州的一家小企业GeneSearch Inc.与蒙大拿州立大学的胚胎学家/分子遗传学家达成的一项合作研究协议。这个第一阶段STTR项目的目标是在一年内准备好Mouse Dracula工具，以进入第二阶段面向市场的开发。

DESCRIPTION (provided by applicant): Procedures on pre-implantation embryos are a cornerstone of much biomedical research; however in all cases, the procedures are very difficult and delicate, requiring exceptionally skilled technical experts and expensive equipment. The difficulties associated with these procedures often results in most such work being avoided by most biomedical research groups. When it must be performed, it is generally out-sourced to dedicated laboratories or facilities. Two downsides to this are: (1) many important questions are likely avoided due to the cost and difficulty of these procedures; and (2) many unique and potentially valuable mouse lines are likely lost or abandoned because the cost and/or complexity of cryopreservation are prohibitive. GeneSearch, Inc. has developed a novel co-axial embryo manipulation tool that promises to address these issues. The innovative elegance of this tool lies in its simple design, in which, rather than having separate "holding" and "manipulating" pipettes that work at opposite poles of the embryo, the Dracula has the manipulating pipette within the holding pipette. The major advantage is that the manipulating pipette contacts the embryo in a region that is constrained by the regulated forces imparted by the holding pipette. Original prototypes of the tool allowed, for the first time, dramatic advances in cryopreservation of large hatched embryos such as those of the llama and horse. Now, GeneSearch is scaling down the size of the tool, using new materials for some parts, and improving its handling specifically for manipulation of mouse-sized embryos. Phase I of the proposed study aims to optimize the "Mouse Dracula" tool for mouse embryological procedures including embryo cryopreservation, production of chimeric mice by ES cell injection, and survival-biopsy for genetic analyses. To accomplish this, we have proposed three specific aims, which (1) optimize tool performance, (2) develop improved protocols for mouse embryo cryopreservation, and (3) test the performance of the tool for chimera production and survival-biopsy-based genetic analyses. This is a collaborative research agreement between a Montana-based small business, GeneSearch Inc., and an embryologist/molecular geneticist at Montana State University. The goal of this Phase I STTR project is to prepare the Mouse Dracula tool to move on to Phase II market-oriented development within a one-year period.