Mouse Stem Cell Research Using the Dracula Pipette

使用 Dracula 移液器进行小鼠干细胞研究

• 批准号: 9456912
• 负责人: Paul Jon Taylor
• 金额: $ 49.46万
• 依托单位: GENESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-15 至 2019-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9456912
• 关键词: Achievement; Address; Advanced Development; Biomedical Research; Biopsy; Caliber; Cells; Chimera organism; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Consultations; Contracts; Cryopreservation; Development; Embryo; Embryology; Ensure; Epigenetic Process; Evaluation; Feedback; Flushing; Fostering; Freezing; Future; Gene Expression; Genetic; Glass; Hand; Human; Individual; Injectable; Injection of therapeutic agent; Inner Cell Mass; Laboratories; Laboratory Research; Lasers; Legal patent; Licensing; Liquid substance; Llama; Marketing; Messenger RNA; Metals; Methods; Microscope; Modification; Motion; Mus; Outsourcing; Performance; Phase; Positioning Attribute; Procedures; Production; Protocols documentation; Recombinants; Refractory; Reproduction; Secure; Small RNA; Stem Cell Research; Stem cells; Survival Rate; System; Technology; Testing; Time; Work; base; blastocyst; commercialization; cost; design; embryonic stem cell; empowered; genetic analysis; genetic approach; genetic disorder diagnosis; improved; innovation; instrument; micromanipulator; new technology; novel; preimplantation; pup; research and development; tool; transcription factor; user-friendly

Advanced approaches for genetic or epigenetic modification of embryonic stem (ES) cells, including TALEN-, ZFN-, or CRISPR-catalyzed genetic modifications or modulation of gene expression via small RNAs, synthetic mRNAs, or recombinant transcription factors, have revolutionized our ability to modify the cells from which novel mouse lines are produced. In contrast, procedures for utilizing these cells within the context of a recipient embryo have changed little in decades. Most embryological procedures require skilled expertise to perform; some, e.g. those involving hatched or manipulated blastocysts, are inaccessible to even expert embryologists. We have developed a novel co-axial embryo manipulation tool, the “Mouse Dracula Pipette”, which addresses these issues. This innovative tool is based on an elegant patented concept in which, rather than having separate “holding” and “manipulating” pipettes that work at opposite poles of the embryo, the manipulating pipette is contained within the holding pipette. The major advantage is that the manipulating pipette contacts the embryo in a region that is constrained under the regulated forces imparted by the holding pipette. The original Dracula Pipette was conceived and developed by GeneSearch, Inc. for use with llama blastocysts, which are ~1 mm in diameter and had been impossible to cryopreserve. By facilitating replacement of blastocoel fluid with cryoprotectant, and flushing of this at thawing, this tool allowed the first reliable freezing and post-thaw reanimation of llama embryos. Reflecting the size of llama blastocysts, the original Dracula was large, hand-held, and constructed from drawn-glass. More recently we have developed the “Mouse Dracula” for small embryos like those of mice or humans, which are ~103-times smaller in volume than llama blastocysts. Scaling down to this size required high-performance materials, precision machining, and micromanipulator control. The tool was optimized for procedures including general manipulations, cryopreservation, cell injection, and biopsy of trophectoderm cells of mouse blastocysts. Advanced materials meld performance with glass-like transparency. Precision linear motion of the injection/biopsy probe is hydraulic, using a metal-bellows. The fine control afforded by the Mouse Dracula makes it effective for harmfree manipulations even on delicate hatched blastocysts. Here we propose a Fast-Track application that will refine the Mouse Dracula for stem cell procedures and aim toward commercialization for these applications. In Phase 1 of this Fast-Track application, we propose three Specific Aims. In Ph1-Aim 1, we will optimize use of the Mouse Dracula to inject ES cells into the blastocoel for production of chimeric mice. In Ph1-Aim 2, we will optimize the tool for harm-free biopsy of cells from the inner cell mass (ICM). In each case, we will test early-, expanded-, and hatched-blastocysts. Evaluation will include assessment of the quality of pups born from the manipulated embryos, as well as the reliability of biopsied materials for transfer into recipient embryos, ex vivo culture, or genetic analyses. Achievement of these performance milestones will mark completion of Phase 1 and initiate commencement of Phase 2. In Phase 2, we propose two Specific Aims. In Ph2-Aim 1, we will further refine “ease of use” and platform-adaptability of the Mouse Dracula to make it optimally convenient for integration into current mouse embryology facilities. In Ph2-Aim 2, we will further refine the Mouse Dracula, within the confines of the performance criteria achieved in the other aims, for more efficient manufacture and more effective commercialization. Thus, we will investigate means of more efficient high-throughput out-sourcing of component production and assembly, and investigate overall manufacturing, marketing, and distribution strategies for delivering the Dracula technology at a favorable cost to mouse embryology laboratories of all sizes. RELEVANCE. Currently, mouse embryological procedures are accessible to only highly skilled technical experts. Also, some embryological stages, e.g., hatched blastocysts, or some procedures, e.g., biopsy of cells from the ICM, are so delicate that, even in skilled hands, they are not accessible. Phase 1 of this proposal optimizes a tool for mouse stem cell-associated embryo manipulations that will make all procedures more accessible to a broader user group, and will facilitate some currently refractory procedures. Phase 2 aims toward optimization of the user-interface and improvements in manufacture that will favor commercialization of this tool for biomedical research applications.

胚胎干细胞（ES）细胞的遗传或表观遗传修饰的先进方法，包括talen-， 通过小RNA，合成 mRNA或重组转录因子已彻底改变了我们修改细胞的能力 产生了新颖的小鼠线。相反，在接收者的背景下使用这些单元的过程 几十年来，胚胎几乎没有改变。大多数胚胎程序都需要熟练的专业知识才能执行； 有些，例如那些孵化或操纵的胚泡的人甚至是专业的胚胎学家无法访问的。 我们已经开发了一种新型的同轴胚胎操纵工具，即“小鼠吸血管”，它解决了 这些问题。这种创新的工具是基于一个优雅的专利概念，在这种概念中，而不是拥有 在胚胎相对的两极工作的单独的“握持”和“操纵”移液器 移液器包含在固定移液器中。主要优点是操纵移液管接触 在固定移液器赋予的受监管力下受约束的区域中的胚胎。 Genesearch，Inc。构思和开发了原始的Dracula Optette，供Llama胚泡， 直径约为1毫米，是不可能冷冻的。通过促进更换 用冷冻保护剂的胚泡液和解冻时的冲洗，该工具允许第一个可靠的冷冻 和骆驼胚胎的后透后复活。反映骆驼胚泡的大小，原始的德古拉是 大型，手持式，并由绘制玻璃制成。最近，我们开发了“小鼠吸血鬼” 对于小胚胎，例如小鼠或人类的胚胎，其体积比骆驼小约103倍 胚泡。缩小到这种尺寸所需的高性能材料，精确加工和 微型操纵器控制。该工具已针对包括一般操作，包括一般操作， 小鼠胚泡的滋养外胚层细胞的冷冻保存，细胞注射和活检。高级材料 与玻璃透明度融合性能。注射/活检探针的精确线性运动为 液压，使用金属骨骼。小鼠Dracula提供的精细控制使其对Harmfree有效 即使在精致的孵化胚泡上进行操作。 在这里，我们提出了一个快速通道的应用 为这些应用提供商业化。在此快速轨道应用程序的第1阶段中，我们提出了三个 具体目标。在pH1-aim 1中，我们将优化使用小鼠吸血鬼将ES细胞注入胚泡 嵌合小鼠的产生。在ph1-aim 2中，我们将优化内部细胞无害活检的工具 细胞质量（ICM）。在每种情况下，我们都将早期测试，扩展和孵化的爆破囊肿。评估将包括 评估从操纵胚胎中诞生的幼崽的质量，以及活检的可靠性 转移到受体胚胎，离体培养或遗传分析的材料。实现这些 绩效里程碑将标记1阶段的完成，并启动第2阶段的开始。 我们提出了两个具体目标。在PH2-AIM 1中，我们将进一步完善“易用性”和平台适应性 小鼠吸血鬼，使其最佳地集成到当前的小鼠胚胎学设施中。在 PH2-AIM 2，我们将在达到的性能标准的范围内进一步完善鼠标吸血鬼 另一个目的是为了更有效的制造和更有效的商业化。那我们将调查 意味着更有效的高通量产生组件生产和组装，并调查 整体制造，营销和分销策略，用于在A 对各种大小的小鼠胚胎学实验室的有利成本。 关联。当前，仅熟练技术的鼠标胚胎程序可以访问 专家。此外，某些胚胎学阶段，例如孵化的胚泡或某些过程，例如细胞活检 从ICM中，非常精致，即使在熟练的手中，它们也无法访问。该提案的第1阶段 优化鼠标干细胞相关的胚胎操作的工具，该工具将使所有过程更加 更广泛的用户组可以访问，并将促进一些当前的难治性程序。第2阶段的目标 旨在优化用户界面和制造业的改进，这将有利于商业化 此用于生物医学研究应用的工具。