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Knockdown of Dnmt1 and Dnmt3a gene expression disrupts preimplantation embryo development through global DNA methylation



DNA methylation is one of the epigenetic mechanisms that plays critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of methylation, and Dnmt3a, for de novo methylation, are gradually erased from the zygote in succeeding stages and then reestablished in the blastocyst. This study was designed to address the vital role of Dnmt1 and Dnmt3a enzymes by silencing their gene expressions in embryonic development in mice.


Groups were (i) control, (ii) Dnmt1-siRNA, (iii) Dnmt3a-siRNA, and (iv) non-targeted (NT) siRNA. Knockdown of Dnmt genes using siRNAs was confirmed by measuring the targeted proteins using Western blot and immunofluorescence. Following knockdown of Dnmt1 and Dnmt3a in zygotes, the developmental competence and global DNA methylation levels were analyzed after 96 h in embryo cultures.


A significant number of embryos arrested at the 2-cell stage or had undergone degeneration in the Dnmt1 and Dnmt3a knocked-down groups. By 3D observations in super-resolution microscopy, we noted that Dnmt1 was exclusively found in juxtanuclear cytoplasm, while the Dnmt3a signal was preferentially localized in the nucleus, both in trophoblasts (TBs) and embryoblasts (EBs). Interestingly, the global DNA methylation level decreased in the Dnmt1 knockdown group, while it increased in the Dnmt3a knockdown group.


Precisely aligned expression of Dnmt genes is highly essential for the fate of an embryo in the early developmental period. Our data indicates that further analysis is mandatory to designate the specific targets of these methylation/demethylation processes in mouse and human preimplantation embryos.

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Three dimensional


5-Methyl cytosine








DNA methyltransferase




Human chorionic gonadotropin


Intracytoplasmic sperm injection




Pregnant mare’s serum gonadotropin


Room temperature


Somatic cell nuclear transfer


Single cell RNA sequencing


Small interfering RNA




Zygotic genome activation


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This study was financially supported by the Ankara University Research Fund 19B0230004, and FU was supported by the YOK 100/2000 scholarship.


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Authors and Affiliations



FU: Designed and performed the experiments, collected data, performed statistical analyses, and wrote the manuscript

OC: Collected data.

AC: Designed and performed the experiments and wrote the manuscript.

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Correspondence to Alp Can.

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The authors declare no competing interests.

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Uysal, F., Cinar, O. & Can, A. Knockdown of Dnmt1 and Dnmt3a gene expression disrupts preimplantation embryo development through global DNA methylation. J Assist Reprod Genet 38, 3135–3144 (2021).

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  • DNA methylation
  • Dnmt1
  • Dnmt3a
  • Embryo
  • siRNA