Abstract
Genetically modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience. Here we present a fully automated system for microinjection into the pronucleus to facilitate genetic modification. We first developed software that automatically controls the microinjection system hardware. The software permits automatic rotation of the zygote to move the pronucleus to the injection pipette insertion position. We also developed software that recognizes the pronucleus in 3-dimensional coordinates so that the injection pipette can be automatically inserted into the pronucleus, and achieved a 94% insertion rate by linking the 2 pieces of software. Next, we determined the optimal solution injection conditions (30 hPa, 0.8–2.0 s) by examining the survival rate of injected zygotes. Finally, we produced transgenic (traditional DNA injection and piggyBac Transposon system) and knock-in (genomic editing) mice using our newly developed Integrated Automated Embryo Manipulation System (IAEMS). We propose that the IAEMS will simplify highly reproducible pronuclear stage zygote microinjection procedures.
Highlights
Modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience
To realize the sequence, we developed hardware that incorporates the electric manipulation system (EMS) and electric injector that we previously developed into a microscope (Fig. 2, Fig. S1)
Existing manual microinjection systems are equipped with a manipulator that moves the pipette in 3-dimensions, a sample stage that moves the zygote in 2 dimensions, and a pump for solution injection
Summary
Modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience. Various methods are used for genetic modification in mice, such as manual injection of a solution into the pronucleus of the mouse zygote. We designed a fully automated method for injecting a solution into the zygote pronucleus that makes genetic modifications by microinjection easy and highly reproducible.
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