Mouse Stem Cell Research Using the Dracula Pipette

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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.