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

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，程序都非常困难和微妙，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避免大多数此类工作。当必须进行时，通常外包给专门的实验室或设施。这有两个缺点：（1）由于这些方法的成本和难度，许多重要的问题可能被避免;和（2）由于冷冻保存的成本和/或复杂性过高，许多独特的和潜在有价值的小鼠品系可能丢失或被放弃。基因搜索公司开发了一种新型的同轴胚胎操作工具，有望解决这些问题。这种工具的创新优雅在于其简单的设计，其中，而不是有单独的“持有”和“操纵”移液管，在胚胎的相反两极工作，德古拉有操纵移液管内的持有移液管。主要优点是操纵移液管在由保持移液管施加的调节力约束的区域中接触胚胎。该工具的原始原型第一次允许戏剧性的进步 冷冻保存大型孵化胚胎，如美洲驼和马的胚胎。现在，GeneSearch正在缩小工具的尺寸，在某些部分使用新材料，并专门针对小鼠大小的胚胎进行操作。拟议研究的第一阶段旨在优化用于小鼠胚胎学程序的“Mouse Dracula”工具，包括胚胎冷冻保存，通过ES细胞注射生产嵌合小鼠，以及用于遗传分析的存活活检。为了实现这一目标，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）测试嵌合体生产和基于存活活检的遗传分析工具的性能。这是一个合作研究协议之间的蒙大拿州的小企业，基因搜索公司，和蒙大拿州立大学的胚胎学家/分子遗传学家。第一阶段STTR项目的目标是在一年的时间内准备Mouse Dracula工具进入第二阶段市场导向的开发。

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.