Acta Veterinaria et Zootechnica Sinica ›› 2022, Vol. 53 ›› Issue (6): 2015-2023.doi: 10.11843/j.issn.0366-6964.2022.06.034

• RESEARCH NOTES • Previous Articles     Next Articles

The Regulation of Methylation Level of IGF2R Gene in IVF Blastocysts Derived from Vitrified Bovine Oocytes by dCas9-SunTag-DNMT3A Technology

YANG Sha1, YANG Yuze2, XU Xi1, HAO Haisheng1, DU Weihua1, PANG Yunwei1, ZHAO Shanjiang1, ZOU Huiying1, ZHU Huabin1, ZHAO Xueming1*   

  1. 1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Beijing General Station of Animal Husbandry, Beijing 100101, China
  • Received:2021-11-16 Online:2022-06-23 Published:2022-06-25

Abstract: The study aimed to explore the effect of dCas9-SunTag-DNMT3A editing system on the methylation level of IGF2R gene and embryonic developmental ability in IVF blastocysts from vitrified bovine oocytes, and to lay the foundation for precise control of DNA methylation at specific sites of vitrified oocytes/embryos. In this study, the in vitro matured bovine oocytes were vitrified, and followed by in vitro fertilization, and the prokaryotic embryos obtained by fertilization were injected with dCas9-SunTag-DNMT3A editing system, and the developmental ability of oocytes were analyzed; the methylation level of the IGF2R gene promoter was detected by sulfite sequencing, and the expression levels of IGF2R and related genes were detected by fluorescence quantitative PCR. Compared with the vitrified group, after injection of different concentrations of dCas9-SunTag-DNMT3A editing system, only the 40 ng·μL-1 group significantly improved the developmental ability of vitrified oocytes after IVF (P < 0.05). There was no significant difference between the 20 and the 60 ng·μL-1 groups (P > 0.05), but the developmental effect of the 40 ng·μL-1 group was still significantly lower than that of the fresh control group (P < 0.05). The methylation level of IGF2R gene promoter in the 40 ng·μL-1 group was found to be similar to that of the fresh group and significantly higher than that of the vitrified group (P < 0.05). The mRNA level of IGF2R gene in the 40 ng·μL-1 group was significantly lower than that in the vitrified group (P < 0.05), which was similar to the fresh group. Injection of 40 ng·μL-1 dCas9-SunTag-DNMT3A methylation editing system can effectively increase the methylation level of IGF2R gene promoter (P < 0.05) and significantly reduce its mRNA expression level (P < 0.05), to positively regulate the development of IVF embryos derived from vitrified oocyte, so that the cleavage rate and blastocyst rate were significantly increased (P < 0.05), and the expression of embryonic development-related genes was promoted.

Key words: dCas9-SunTag-DNMT3A, vitrification, bovine, IGF2R

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