Bovine embryos were surgically collected from the oviducts of superovulated crossbred heifers 48 h postonset of estrus. The one-celled ova were treated with 4'-6'-diamidino-2-phenylindole (DAPI) and observed under ultraviolet light by fluorescence microscopy. Both male and female pronuclei were visualized, identified and subjected to micromanipulation.
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Importance of nuclear localization signal-fused Cas9 in the production of genome-edited mice via embryo electroporation.
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Division of Science and Engineering, Graduate School of Arts and Science, Iwate University, Iwate, 020-8551, Japan; Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Iwate, 020-8551, Japan. Electronic address:
Previously, to generate genome-edited animals by introducing CRISPR-associated protein 9 (Cas9) into embryos, we developed the Technique for Animal Knockout system by Electroporation (TAKE). Additionally, by fluorescently labeling Cas9, we successfully visualized the Cas9 introduced into the pronuclei of embryos; however, whether Cas9 was introduced directly into the pronuclei by electric pulse or transferred from the cytoplasm by nuclear localization signal (NLS) remained unknown. Herein, we evaluated the localization of Cas9 with (Cas9-NLS) or without NLS (Cas9-noNLS) in mice embryos following electroporation by fusing them with GFP.
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Theriogenology
April 2023
Department of Biological Production, Tokyo University of Agriculture and Technology, Tokyo, 183-8538, Japan. Electronic address:
Article Synopsis
- Direct cleavage is an abnormal cleavage process in which a zygote divides into three or more cells, more common in those with multiple pronuclei (multi-PN) and abnormal pronuclei migration.
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