In JoVE (1)
Other Publications (1)
Articles by Masatoshi Ooga in JoVE
Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development Masatoshi Ooga1,3, Satoshi Funaya2, Fugaku Aoki2, Teruhiko Wakayama1,3 1Faculty of Life and Environmental Sciences, Department of Biotechnology, University of Yamanashi, 2Department of Integrated Bioscience, Graduate School of Frontier Sciences, University of Tokyo, 3Advanced Biotechnology Center, University of Yamanashi Chromatin looseness appears to be involved in the developmental potential of blastomeres. However, it is not known whether chromatin looseness can be used as a reliable index for the developmental potential for embryos. Here, an experimental system in which chromatin looseness-evaluated zygotes can develop to full term has been described.
Other articles by Masatoshi Ooga on PubMed
FRAP Analysis of Chromatin Looseness in Mouse Zygotes That Allows Full-term Development PloS One. 2017 | Pubmed ID: 28542635 Chromatin looseness, which can be analyzed by fluorescence recovery after photobleaching (FRAP) using eGFP-tagged core histone proteins, is an important index of the differentiation potential of blastomere cells and embryonic stem cells. Whether chromatin looseness is a reliable index of the developmental potential of embryos during ontogenesis is not known. As a necessary first step toward answering this question, we investigated whether FRAP-analyzed embryos are capable of normal preimplantation and full-term development. All tested concentrations (50, 100, and 250 ng/μL) of microinjected eGFP-H2B mRNA were sufficient for detecting differences in chromatin looseness between male and female pronuclei. After FRAP analysis, most of the zygotes developed into blastocysts. Importantly, a considerable number of offspring developed from the FRAP analyzed zygotes (32/78; 41.0%) and grew into healthy adults. The offspring of zygotes injected with 250 ng/μL of eGFP-H2B mRNA and bleached using 110 μW laser power for 5 s were not genetically modified. Interestingly, bleaching using a 3-fold stronger laser intensity for a 6-fold longer time did not cause toxicity during preimplantation development, indicating that bleaching did not critically affect preimplantation development. Finally, we confirmed that similar results were obtained using two different types of confocal laser-scanning microscopes. This FRAP protocol would be useful for investigating the association between chromatin looseness and development.