Gene Silencing of DNA Methyltransferases by RNA Interference in Bovine Fibroblast Cells

Yamanaka, Kenichi; Balboula, Ahmed Z.; Sakatani, Miki; Takahashi, Masashi

doi:10.1262/jrd.09-105a

Cited by 10 publications

(7 citation statements)

References 48 publications

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“…Then, expression of the gene would gradually recover. mRNA and protein degeneration was detected at 48 and 96 h after siRNA transfection, which are the time points believed to be the most appropriate for detecting bovine fibroblast cell RNA silencing based on previous studies (Yamanaka et al. 2010).…”

Section: Discussionmentioning

confidence: 93%

Histone Deacetylase 1 Down‐Regulation on Developmental Capability and Histone Acetylation in Bovine Oocytes and Parthenogenetic Embryos

Zhong

Zhao

Zhang

et al. 2011

Reprod Domestic Animals

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Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells, including bovine oocytes and pre-implantation embryos. However, the biological functions of HDAC1 in supporting the growth and development of bovine oocytes and embryos are still not fully elucidates. In this study, three siRNAs (si299, si672, and si1272) targeting to HDAC1 mRNA sequence were designed. After transfection into bovine fibroblast cells, si299, the most effective one in HDAC1 knock-down, was selected. The selected siRNA was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDAC1 in the maturation of bovine oocytes. Finally, the siRNA was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. The rates of cleavage, blastocyst development and acetylation of lysine 14 of H3 (H3K14) state were checked. The results suggest that HDAC1 knock-down in oocytes did not influence the rates of maturation or cleavage of parthenogenetic embryos. However, the rates of blastocyst decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation level increased after siRNA microinjection into parthenogenetic embryos.

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Section: Discussionmentioning

confidence: 93%

Histone Deacetylase 1 Down‐Regulation on Developmental Capability and Histone Acetylation in Bovine Oocytes and Parthenogenetic Embryos

Zhong

Zhao

Zhang

et al. 2011

Reprod Domestic Animals

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“…Bovine fibroblast cells were obtained from the ear of a 20‐day‐old Japanese Black male calf and used as donor cells. Preparation procedure was performed as described previously (Yamanaka et al . 2010).…”

Section: Methodsmentioning

confidence: 99%

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Yamanaka

Kaneda

Inaba

et al. 2011

Animal Science Journal

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Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.

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“…The siRNA were dissolved with RNase free H2O according to the manufacturer's instructions. The specificity of the siRNA for knockdown of DNMT1 was confirmed in bovine fibroblast cells, not the knockdown effect against other DNMTs mRNA [29]. After activation, the embryos were transferred in a 20-μl drop of CR1aa supplemented with 5% (v/v) FCS for microinjection.…”

Section: Design Of Sirna Design and Microinjection Into Embryosmentioning

confidence: 99%

Effects of Downregulating DNA Methyltransferase 1 Transcript by RNA Interference on DNA Methylation Status of the Satellite I Region and In Vitro Development of Bovine Somatic Cell Nuclear Transfer Embryos

Yamanaka

Sakatani

Kubota

et al. 2011

Journal of Reproduction and Development

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Abstract. For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency. Key words: DNA methylation, DNA methyltransferase, Embryo development, Nuclear transfer, RNA interference (J. Reprod. Dev. 57: [393][394][395][396][397][398][399][400][401][402] 2011) hile there have been many attempts to improve the developmental competence of somatic cell nuclear transfer (NT) embryos, the success rate of producing viable offspring is still very low, with less than 5% of embryos transferred to surrogate females [1]. In bovine cloning, high rates of embryonic, fetal, neonatal and postnatal abnormalities have been consistently observed [2][3][4]. To achieve developmental competence of NT embryos, the donor nucleus of the differentiated cell needs to undergo epigenetic reprogramming during preimplantation development. However, this process needed for a differentiated donor nucleus to achieve embryonic status is delayed and incomplete in NT embryos [5]. In fact, many studies have demonstrated that epigenetic statuses such as DNA methylation and histone acetylation, are abnormally established in NT embryos during preimplantation development [6][7][8][9][10][11][12]. As a result, abnormal gene expression including imprinted genes [10,[13][14][15] can prevent normal development of NT embryos [16].DNA methylation of cytosine residues in CpG dinucleotides is one of the epigenetic modifications mediated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3a and DNMT3b [17]. DNA methylation patterns are dynamic, yet tightly regulated, during mammalian development [18]. In normal embryos, both maternal and paternal gametes undergo extensive demethylation after fertilization, with the paternal genome undergoing demethylation within hours of fertilization [19]. In contrast, the maternal genome is passively...

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Gene Silencing of DNA Methyltransferases by RNA Interference in Bovine Fibroblast Cells

Cited by 10 publications

References 48 publications

Histone Deacetylase 1 Down‐Regulation on Developmental Capability and Histone Acetylation in Bovine Oocytes and Parthenogenetic Embryos

Histone Deacetylase 1 Down‐Regulation on Developmental Capability and Histone Acetylation in Bovine Oocytes and Parthenogenetic Embryos

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Effects of Downregulating DNA Methyltransferase 1 Transcript by RNA Interference on DNA Methylation Status of the Satellite I Region and In Vitro Development of Bovine Somatic Cell Nuclear Transfer Embryos

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