Mouse Stem Cell Research Using the Dracula Pipette

胚胎干细胞的遗传或表观遗传修饰的先进方法，包括TALEN-， ZFN或CRISPR催化的遗传修饰或通过小RNA调节基因表达，合成 MRNAs，或重组转录因子，已经彻底改变了我们修改细胞的能力 产生了新的小鼠品系。相反，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才能进行； 有些胚胎，例如那些涉及孵化或操纵的囊胚，即使是专家胚胎学家也无法接触到。 我们开发了一种新型的同轴胚胎处理工具--“小鼠德古拉吸管”，它解决了 这些问题。这一创新的工具基于一个优雅的专利概念，其中没有 在胚胎的相反两极工作的单独的“握持”和“操纵”吸管， 吸液管容纳在保持吸液管内。主要的优点是操作吸量管接触 在固定吸管所施加的规则力的约束下的区域中的胚胎。这个 最初的德古拉吸管是由GeneSearch，Inc.构思和开发的，用于骆驼胚泡， 它们的直径约为1 mm，无法超低温保存。通过促进更换 胚胎液中含有冷冻保护剂，并在解冻时冲洗，这一工具允许第一次可靠的冷冻 以及骆驼胚胎解冻后的复活。反映出骆驼胚泡的大小，最初的德古拉是 大的，手持的，用拉丝玻璃建造的。最近，我们开发出了“老鼠德古拉” 对于像老鼠或人类这样的小胚胎来说，它们的体积大约是骆驼的103倍 胚泡。缩小到这种尺寸需要高性能材料、精密加工和 微操作器控制。该工具针对包括常规操作在内的程序进行了优化， 小鼠胚泡滋养外胚层细胞的冷冻保存、细胞注射和活检。先进材料 将性能与玻璃般的透明度融合在一起。注射/活组织检查探头的精确直线运动 液压，使用金属波纹管。老鼠德古拉提供的精细控制使其有效地无害 即使是在精致的孵化胚泡上也是如此。 在这里，我们提出了一个快速跟踪应用程序，它将完善小鼠Dracula的干细胞程序和目标 走向这些应用的商业化。在此Fast-Track应用程序的第一阶段，我们提出三个 明确的目标。在PH1-AIM 1中，我们将优化使用小鼠德拉库拉将ES细胞注射到胚泡中 生产嵌合体小鼠。在PH1-AIM 2中，我们将优化从内部对细胞进行无害化活组织检查的工具 细胞质量(ICM)。在每种情况下，我们都将测试早期、扩大和孵化的囊胚。评估将包括 评估从操纵胚胎中出生的幼崽的质量以及活组织检查的可靠性 移植到受体胚胎、体外培养或遗传分析的材料。实现这些目标 绩效里程碑将标志着第一阶段的完成，并启动第二阶段的开始。在第二阶段， 我们提出了两个具体目标。在PH2-AIM 1中，我们将进一步细化 Mouse Dracula，使其能够以最佳方式方便地集成到当前的小鼠胚胎设施中。在……里面 PH2-目标2，我们将在以下性能标准的范围内进一步改进Mouse Dracula 另一个目标是更高效的制造和更有效的商业化。因此，我们将调查 更高效、高产量地外包零部件生产和组装的方法，并调查 全面的制造、营销和分销战略，以便在一年内交付Dracula技术 对各种规模的小鼠胚胎学实验室来说都是有利的成本。 关联性。目前，只有高度熟练的技术人员才能使用小鼠胚胎程序。 专家。此外，一些胚胎学阶段，如孵化的囊胚，或一些程序，如细胞活组织检查 来自ICM的，是如此脆弱，即使在熟练的人手中，也是无法接触到的。此提案的第一阶段 优化了一种用于小鼠干细胞相关胚胎操作的工具，将使所有程序 更广泛的用户群体可以访问，并将促进一些目前难以完成的程序。第二阶段目标 用户界面的优化和制造的改进将有利于商业化 这种工具适用于生物医学研究。

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.