Abstract
To understand the spatiotemporal changes in cellular status that occur during embryonic development, it is desirable to detect simultaneously the expression of genes, proteins, and epigenetic modifications in individual embryonic cells. A technique termed methylation-specific fluorescence in situ hybridization (MeFISH) was developed recently that can visualize the methylation status of specific DNA sequences in cells fixed on a glass slide. Here, we adapted this glass slide-based MeFISH to the study of intact embryos, and established a method called whole-mount MeFISH. This method can be applied to any DNA sequences in theory and, as a proof-of-concept experiment, we examined the DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, and obtained a result that was consistent with previous findings, thus validating the MeFISH method. We also succeeded in combining whole-mount MeFISH with immunostaining or RNA fluorescence in situ hybridization (RNA-FISH) techniques by adopting steps to retain signals of RNA-FISH or immunostaining after harsh denaturation step of MeFISH. The combined methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution.
| Original language | English |
|---|---|
| Article number | e95750 |
| Journal | PLoS One |
| Volume | 9 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Apr 22 2014 |
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All Science Journal Classification (ASJC) codes
- Agricultural and Biological Sciences(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Medicine(all)
Cite this
Whole-mount MeFISH : A novel technique for simultaneous visualization of specific DNA methylation and protein/RNA expression. / Shiura, Hirosuke; Okamoto, Akimitsu; Sasaki, Hiroyuki; Abe, Kuniya.
In: PLoS One, Vol. 9, No. 4, e95750, 22.04.2014.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Whole-mount MeFISH
T2 - A novel technique for simultaneous visualization of specific DNA methylation and protein/RNA expression
AU - Shiura, Hirosuke
AU - Okamoto, Akimitsu
AU - Sasaki, Hiroyuki
AU - Abe, Kuniya
PY - 2014/4/22
Y1 - 2014/4/22
N2 - To understand the spatiotemporal changes in cellular status that occur during embryonic development, it is desirable to detect simultaneously the expression of genes, proteins, and epigenetic modifications in individual embryonic cells. A technique termed methylation-specific fluorescence in situ hybridization (MeFISH) was developed recently that can visualize the methylation status of specific DNA sequences in cells fixed on a glass slide. Here, we adapted this glass slide-based MeFISH to the study of intact embryos, and established a method called whole-mount MeFISH. This method can be applied to any DNA sequences in theory and, as a proof-of-concept experiment, we examined the DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, and obtained a result that was consistent with previous findings, thus validating the MeFISH method. We also succeeded in combining whole-mount MeFISH with immunostaining or RNA fluorescence in situ hybridization (RNA-FISH) techniques by adopting steps to retain signals of RNA-FISH or immunostaining after harsh denaturation step of MeFISH. The combined methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution.
AB - To understand the spatiotemporal changes in cellular status that occur during embryonic development, it is desirable to detect simultaneously the expression of genes, proteins, and epigenetic modifications in individual embryonic cells. A technique termed methylation-specific fluorescence in situ hybridization (MeFISH) was developed recently that can visualize the methylation status of specific DNA sequences in cells fixed on a glass slide. Here, we adapted this glass slide-based MeFISH to the study of intact embryos, and established a method called whole-mount MeFISH. This method can be applied to any DNA sequences in theory and, as a proof-of-concept experiment, we examined the DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, and obtained a result that was consistent with previous findings, thus validating the MeFISH method. We also succeeded in combining whole-mount MeFISH with immunostaining or RNA fluorescence in situ hybridization (RNA-FISH) techniques by adopting steps to retain signals of RNA-FISH or immunostaining after harsh denaturation step of MeFISH. The combined methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution.
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UR - http://www.scopus.com/inward/citedby.url?scp=84899708165&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0095750
DO - 10.1371/journal.pone.0095750
M3 - Article
C2 - 24755742
AN - SCOPUS:84899708165
VL - 9
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 4
M1 - e95750
ER -