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

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 areprohibitive. 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 manipulatingpipettes 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. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Existing methods of mouse embryomanipulation, while allowing generation of chimeric mice for the production of novel genetically modified lines and allowing embryo cryopreservation for long-term maintenance of inactive mouse lines, are highly technically challenging. This, combined withthe high cost of contracting these services, frequently puts these technologies out of reach for most biomedical research labs. A novel embryo manipulation tool, the Dracula Pipette, was developed in recent years by GeneSearch, Inc., for cryopreservationof llama and horse embryos. GeneSearch has now developed prototypes optimized for use with mouse embryos. It is anticipated that this new tool, the Mouse Dracula , will provide an affordable user-friendly system that will empower even the smallest embryology or reproduction labs to generate chimeric mice and cryopreserve embryos. The proposed STTR Phase I project is aimed at final refinement of this tool for mouse embryology and development of efficient protocols based on this tool for mouse embryo cryopreservation and generation of chimeric mice. We anticipate this will be a very beneficial instrument for many biomedical research laboratories. This work will position GeneSearch to enter Phase II, with an aim for production and global distribution of the Mouse Dracula in the near future.