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Establishment of an integrated automated embryonic manipulation system for producing genetically modified mice.

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.

SUBMITTER: Eto T

PROVIDER: S-EPMC8175685 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge.We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (ΦPSII). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of ΦPSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency.The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

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Establishment of an integrated automated embryonic manipulation system for producing genetically modified mice.

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