RNA-FISH and Immunofluorescence of Mouse Preimplantation and Postimplantation Embryos
There are two modes of X chromosome inactivation (XCI) in the mouse. One mode is imprinted XCI: it is initiated at around the four-cell stage in favor of the paternal X chromosome, and is maintained in the extraembryonic tissues. The other mode is random XCI, which takes place in the epiblast lineage at the periimplantation stage. X-linked noncoding Xist RNA, which becomes upregulated on the X chromosome to be inactivated at the onset of XCI and plays a critical role in both imprinted and random XCI, and its accumulation in the nucleus have been referred to as one of the hallmarks of the presence of the inactivated X chromosome. RNA-FISH has therefore been an invaluable method for the study of XCI. As XCI status changes dynamically during periimplantation development in the mouse, analysis using samples from these developmental stages is absolutely necessary for elucidation of the molecular basis of XCI mechanisms. However, dissection of the embryos at around the periimplantation stages is not easy, and this impedes in vivo analysis of the kinetics of XCI. Here, we describe our methods for dissecting the periimplantation stage embryo and subsequent procedures for RNA-FISH and immunostaining.
Key wordsRNA-FISH Immunofluorescence Preimplantation and postimplantation embryo
We are grateful to Michihiko Sugimoto for technical advice on mouse embryo manipulation, whole-mount 3D RNA-FISH experiments, and developing a method for a strand-specific RNA-FISH probe, and to Osamu Masui for assessment of RNA-FISH probe quality. This work was partly supported by Grants-in-Aid for Scientific Research on Innovative Areas (16H01320 and 17H05606 to TS) and a Grant-in-Aid for Scientific Research (A) (17H01588 to TS) from the Ministry of Educations, Sciences, Sports, and Culture of Japan (MEXT) and Japan Society for the Promotion of Science (JSPS), respectively.
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