Protocol | Published:

Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos

Nature Protocols volume 6, pages 270284 (2011) | Download Citation

Abstract

Dynamic reprogramming of the genome takes place during the gamete-to-embryo transition. This transition defines a period of continuous and global change but has been difficult to study because of extremely limited material and varying degrees of chromatin compaction. Improved methods of detecting chromatin and gene expression changes in the germ line and in the preimplantation embryo would greatly enhance the understanding of this crucial developmental transition. Here we describe a protocol developed and used by us that improves the sensitivity of existing fluorescence in situ hybridization (FISH) methods; the protocol described here has enabled us to visualize single-copy DNA targets and corresponding nascent RNA transcripts in preimplantation embryos and during spermatogenesis. Major improvements over alternative methods involve fixation and permeabilization steps. Chromatin epitopes can be visualized simultaneously by combining FISH with immunofluorescence; multicopy and repetitive element expression can also be reliably measured. This procedure (sample preparation and staining) requires 1–1.5 d to complete and will facilitate detailed examination of spatial relationships between chromatin epitopes, DNA and RNA during the dynamic transition from gamete to embryo.

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Acknowledgements

We thank K. Hyunh, B. Payer and L.-F. Zhang for their assistance to optimize experimental conditions during the initial phases of these works. The studies have been supported by National Institutes of Health grant RO1-GM58839 to J.T.L. S.H.N. was supported by the research fellowships of the Japan Society for the Promotion of Science (JSPS) and the Charles King Trust. J.T.L. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Satoshi H Namekawa

    Present address: Division of Reproductive Sciences, Department of Pediatrics, Perinatal Institute, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.

Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Howard Hughes Medical Institute, Boston, Massachusetts, USA.

    • Satoshi H Namekawa
    •  & Jeannie T Lee
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Satoshi H Namekawa
    •  & Jeannie T Lee

Authors

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Contributions

S.H.N. and J.T.L. wrote the manuscript. S.H.N. designed the protocols, with guidance from lab protocols, and J.T.L. and S.H.N. conducted the experiments. J.T.L. supervised the projects.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Satoshi H Namekawa or Jeannie T Lee.

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DOI

https://doi.org/10.1038/nprot.2010.195

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