Mouse Stem Cell Research Using the Dracula Pipette

Advanced approaches for genetic or epigenetic modification of embryonic stemEScellsincluding TALENZFNor CRISPR catalyzed genetic modifications or modulation of gene expression via small RNAssynthetic mRNAsor recombinant transcription factorshave revolutionized our ability to modify the cells from which novel mouse lines are producedIn contrastprocedures for utilizing these cells within the context of a recipient embryo have changed little in decadesMost embryological procedures require skilled expertise to performsomee gthose involving hatched or manipulated blastocystsare inaccessible to even expert embryologistsWe have developed a novel co axial embryo manipulation tooltheMouse Dracula Pipettewhich addresses these issuesThis innovative tool is based on an elegant patented concept in whichrather than having separateholdingandmanipulatingpipettes that work at opposite poles of the embryothe manipulating pipette is contained within the holding pipetteThe major advantage is that the manipulating pipette contacts the embryo in a region that is constrained under the regulated forces imparted by the holding pipetteThe original Dracula Pipette was conceived and developed by GeneSearchIncfor use with llama blastocystswhich aremm in diameter and had been impossible to cryopreserveBy facilitating replacement of blastocoel fluid with cryoprotectantand flushing of this at thawingthis tool allowed the first reliable freezing and post thaw reanimation of llama embryosReflecting the size of llama blastocyststhe original Dracula was largehand heldand constructed from drawn glassMore recently we have developed theMouse Draculafor small embryos like those of mice or humanswhich aretimes smaller in volume than llama blastocystsScaling down to this size required high performance materialsprecision machiningand micromanipulator controlThe tool was optimized for procedures including general manipulationscryopreservationcell injectionand biopsy of trophectoderm cells of mouse blastocystsAdvanced materials meld performance with glass like transparencyPrecision linear motion of the injection biopsy probe is hydraulicusing a metal bellowsThe fine control afforded by the Mouse Dracula makes it effective for harmfree manipulations even on delicate hatched blastocystsHere we propose a Fast Track application that will refine the Mouse Dracula for stem cell procedures and aim toward commercialization for these applicationsIn Phaseof this Fast Track applicationwe propose three Specific AimsIn PhAimwe will optimize use of the Mouse Dracula to inject ES cells into the blastocoel for production of chimeric miceIn PhAimwe will optimize the tool for harm free biopsy of cells from the inner cell massICMIn each casewe will test earlyexpandedand hatched blastocystsEvaluation will include assessment of the quality of pups born from the manipulated embryosas well as the reliability of biopsied materials for transfer into recipient embryosex vivo cultureor genetic analysesAchievement of these performance milestones will mark completion of Phaseand initiate commencement of PhaseIn Phasewe propose two Specific AimsIn PhAimwe will further refineease of useand platform adaptability of the Mouse Dracula to make it optimally convenient for integration into current mouse embryology facilitiesIn PhAimwe will further refine the Mouse Draculawithin the confines of the performance criteria achieved in the other aimsfor more efficient manufacture and more effective commercializationThuswe will investigate means of more efficient high throughput out sourcing of component production and assemblyand investigate overall manufacturingmarketingand distribution strategies for delivering the Dracula technology at a favorable cost to mouse embryology laboratories of all sizesRELEVANCECurrentlymouse embryological procedures are accessible to only highly skilled technical expertsAlsosome embryological stagese ghatched blastocystsor some procedurese gbiopsy of cells from the ICMare so delicate thateven in skilled handsthey are not accessiblePhaseof this proposal optimizes a tool for mouse stem cell associated embryo manipulations that will make all procedures more accessible to a broader user groupand will facilitate some currently refractory proceduresPhaseaims toward optimization of the user interface and improvements in manufacture that will favor commercialization of this tool for biomedical research applications While technologies for modifying stem cells in culture have advanced in recent yearstechnologies for using these cells in recipient embryos have not advanced substantiallyExisting methods are technically challengingcannot effectively achieve reliable harm free stem cell biopsy and are not practical for use on late stagehatchedblastocystsThusmany embryological technologies are out of reach for most small or unspecialized biomedical research labsand some important conceivable manipulations are out of reach for all laboratoriesGeneSearchIncpreviously developed a novel embryo manipulation tooltheDracula Pipettefor cryopreservation of llama embryos and patented the innovative coaxial design of the Dracula technologyWe recently developed an advanced version of the Dracula optimized for use with the much smaller embryos of mice or humansTheMouse Draculawill provide an affordable user friendly system that will empower a much larger number of embryology or reproduction labs to generate chimeric mice or recover stem cells from early or late stage blastocystsIn additionwe anticipate that the delicate manipulations afforded by this tool will empower new manipulations that cannot be reasonably achieved by contemporary approachesIn addition to optimizing this tool and protocols using it for stem cell procedures on earlyand late stage mouse blastocysts within Phasethis Fast track proposal includes Phasetechnological refinements aimed at optimizing the user interface and better positioning the tool for future commercial productionand a Commercialization Plan that extends beyond this