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.

用于胚胎干细胞（ES）的遗传或表观遗传修饰的先进方法，包括TALEN-， ZFN或CRISPR催化的遗传修饰或经由小RNA、合成RNA或合成RNA调节基因表达 mRNAs，或重组转录因子，已经彻底改变了我们修饰细胞的能力， 产生新的鼠标线。相比之下，在接受者的环境中利用这些细胞的程序 几十年来胚胎几乎没有变化。大多数胚胎学程序需要熟练的专业知识来执行; 有些方法，例如涉及孵化或操作的胚泡的方法，甚至胚胎学家专家也无法获得。 我们已经开发了一种新型的同轴胚胎操作工具，“小鼠德古拉吸管”，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，而不是有 分离的“保持”和“操纵”移液管在胚胎的相反两极工作， 移液管包含在保持移液管内。主要优点是操作移液管接触 胚胎在由保持移液管施加的调节力下被约束的区域中。的 最初的Dracula移液器是由GeneSearch，Inc.为了用于美洲驼胚泡， 其直径约为1 mm，不可能冷冻保存。通过促进更换 囊胚腔液与冷冻保护剂，并冲洗这在解冻，这个工具允许第一个可靠的冷冻 和美洲驼胚胎解冻后的复苏。反映了美洲驼囊胚的大小，最初的德古拉是 大的，手持的，由拉制玻璃制成的。最近，我们开发了“老鼠德古拉” 对于小鼠或人类的小胚胎来说，它们的体积比美洲驼小103倍。 囊胚缩小到这个尺寸需要高性能材料，精密加工， 显微操作器控制该工具针对包括一般操作在内的程序进行了优化， 小鼠胚泡的滋养外胚层细胞的冷冻保存、细胞注射和活组织检查。先进材料 将性能与玻璃般的透明度融为一体。注射/活检探头的精确线性运动是 液压的，用金属波纹管德古拉老鼠提供的精细控制使其有效无害 即使是在脆弱的孵化胚泡上。 在这里，我们提出了一个快速通道应用程序，将完善小鼠德古拉干细胞程序和目标 这些应用的商业化。在此快速通道应用程序的第1阶段，我们提出了三个 具体目标。在Ph 1-Aim 1中，我们将优化使用小鼠Dracula将ES细胞注射到囊胚腔中， 嵌合小鼠的产生。在Ph 1-Aim 2中，我们将优化工具，从内部对细胞进行无害活检 细胞团（ICM）。在每种情况下，我们将测试早期，扩展和孵化囊胚。评价将包括 评估从操作胚胎出生的幼崽的质量，以及活检的可靠性， 用于转移到受体胚胎、离体培养或遗传分析的材料。实现这些 性能里程碑将标志着第1阶段的完成，并启动第2阶段的开始。在第二阶段， 我们提出两个具体目标。在Ph 2-Aim 1中，我们将进一步完善“易用性”和平台适应性， 小鼠德古拉，使其最佳方便整合到目前的小鼠胚胎学设施。在 Ph 2-目标2，我们将进一步完善老鼠德古拉，在性能标准的范围内实现， 另一个目标是更有效的生产和更有效的商业化。因此，我们将调查 部件生产和组装的更有效的高吞吐量外包手段，并调查 全面的制造，营销和分销战略，以提供德古拉技术在一个 对所有规模的小鼠胚胎学实验室来说，成本都是有利的。 本案无关目前，只有高度熟练的技术人员才能进行小鼠胚胎学程序。 专家此外，一些胚胎学阶段，例如，孵化胚泡，或一些程序，例如，细胞活组织检查 来自ICM，是如此的脆弱，即使在熟练的手中，他们也无法接近。本提案的第一阶段 优化了一种用于小鼠干细胞相关胚胎操作的工具， 更广泛的用户群可以访问，并将促进一些目前难治的程序。第二阶段目标 朝着优化用户界面和改进制造，这将有利于商业化 这是一个生物医学研究应用的工具。