Aug 26, 2025
Construction of protein-based artificial kinetochore in mouse oocytes
- Yuanzhuo Zhou1,2,
- Kohei Asai1,2,
- Hirohisa Kyogoku1,3,
- Tomoya S. Kitajima1,2
- 1Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan;
- 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan;
- 3Graduate School of Agricultural Science, Kobe University, Kobe, Japan
- Designing protein-based artificial kinetochore
External link: https://doi.org/10.1038/s41556-025-01792-w
Protocol Citation: Yuanzhuo Zhou, Kohei Asai, Hirohisa Kyogoku, Tomoya S. Kitajima 2025. Construction of protein-based artificial kinetochore in mouse oocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw41k9lmk/v1
Manuscript citation:
Zhou, Y., Asai, K., Kyogoku, H. et al. Designing protein-based artificial kinetochores as decoys to prevent meiotic errors in oocytes. Nat Cell Biol (2025). https://doi.org/10.1038/s41556-025-01792-w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 24, 2025
Last Modified: August 26, 2025
Protocol Integer ID: 225314
Keywords: artificial kinetochore in mouse oocyte, based artificial kinetochore, mouse oocyte, construction of protein, protein, mouse
Abstract
This protocol describes the workflow of construction of protein-based artificial kinetochore in mouse oocytes.
Materials
Pregnant mare serum gonadotropin (PMSG); mice (8–12 weeks old); M2 medium; IBMX (Sigma); mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific); mRNAs for NDC80-FKBP, NUF2, H2B-mCherry, hAG-Pfv-Sapphire-GS-FRB, FKBP-GS-FKBP; rapamycin (Funakoshi).
Troubleshooting
Inject mice (8–12 weeks old) with 0.2 ml (10 IU) pregnant mare serum gonadotropin (PMSG).
Collect fully grown GV-stage oocytes 48 hours after PMSG injection.
Incubate oocytes in M2 medium containing 200 μM IBMX (Sigma) at 37 °C.
Transcribe and purify mRNAs using the mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific).
Prepare the mRNA mixture containing 0.3 pg NDC80-FKBP, 0.3 pg NUF2, 0.45 pg H2B-mCherry, 3.5 pg hAG-Pfv-Sapphire-GS-FRB, and 0.15 pg FKBP-GS-FKBP.
Inject the mRNA mixture into GV-arrested oocytes, followed by 2 hours incubation at 37 °C.
Induce meiotic resumption by washing out IBMX.
Culture oocytes in M2 medium containing 500 nM rapamycin (Funakoshi) to induce formation of the protein-based artificial kinetochore (NDC80-GEM-Linker).