Abstract Delay in maternal transcript degradation of DNMT1 (DNA (cytosine-5)-methyltransferase 1cytosine-5)-methyltransferase 1) in porcine (Sus scrofa) somatic cell nuclear transfer embryos compared with in vitro fertilized counterparts was confirmed, thereafter anti-DNMT1 siRNA was injected into embryos at one-cell stage to evaluate the effect of DNMT1 knockdown on the developmental rate of embryos and the mRNA levels and DNA methylation levels of several developmentally important genes. Though a higher proportion of blastocysts was obtained in 50 μmol/L siRNA injected embryos (30.43%) compared with mock injected embryos (23.01%) and untreated controls (25.74%), while the difference was not statistically significant (P= 0.19). In the case of genes expression, inhibition of DNMT1 significantly increased the mRNA levels of POU5F1 at four-cell stage and of NANOG at the blastocyst stage (P＜0.05). In the aspect of DNA methylation, inhibition of DNMT1 reduced the DNA methylation level of POU5F1 at four-cell stage and decreased the general methylation to the level of in vivo produced embryos at the blastocyst stage, whereas over-depressed DNMT1 disrupted the imprinted methylation of a long noncoding RNA H19. This study suggests that mild inhibition of over-expressed DNMT1 facilitates pluripotent genes transcription and demethylation reprogramming of porcine cloned embryos, furthermore provides a scientific reference for further research on function mechanisms of DNMT1 during early embryogenesis.
XU Wei-Hua,LI Zi-Cong,WU Zhen-Fang, et al. Effect of DNMT1 Interference on Development, Gene Transcription and DNA Methylation of Porcine (Sus scrofa) Cloned Embryos[J]. , 2018, 26(12): 2075-2083.
Bauer BK, Isom SC, Spate LD, et al. 2010. Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos[J]. Biology of Reproduction, 83(5):791-798.
Ge SX. 2007. Exploratory bioinformatics investigation reveals importance of "junk" DNA in early embryo development[J]. BMC Genomics, 18(1):200.
Giraldo AM, Vaught TD, Fu L, et al. 2009. Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells[J]. Gene Expression, 14(5):251-263.
Han DW, Im YB, Do JT, et al. 2008. Methylation status of putative differentially methylated regions of porcine IGF2 and H19[J]. Molecular Reproduction and Development, 75(5):777-784.
He S, Sun H, Lin L, et al. 2017. Passive DNA demethylation preferentially up-regulates pluripotency-related genes and facilitates the generation of induced pluripotent stem cells[J]. The Journal of Biological Chemistry, 292(45):18542-18555.
Hirasawa R, Chiba H, Kaneda M, et al. 2008. Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development[J]. Genes & Development, 22(12):1607-1616.
Huan Y, Wang H, Wu Z, et al. 2015a. The expression patterns of DNA methylation reprogramming related genes are associated with the developmental competence of cloned embryos after zygotic genome activation in pigs[J]. Gene Expression Patterns, 18(1-2):1-7.
Huan Y, Wu Z, Zhang J, et al. 2015b. Epigenetic Modification Agents Improve Gene-Specific Methylation Reprogramming in Porcine Cloned Embryos[J]. PLoS One, 10(6):e0129803.
Ju S, Rui R, Lu Q, et al. 2010. Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro[J]. Journal of Assisted Reproduction and Genetics, 27(1):49-59.
Kang YK, Koo DB, Park JS, et al. 2001. Typical demethylation events in cloned pig embryos. Clues on species-specific differences in epigenetic reprogramming of a cloned donor genome[J]. The Journal of Biological Chemistry, 276(43):39980-39984.
Kumar BM, Jin HF, Kim JG, et al. 2007. Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells[J]. Developmental Dynamics, 236(2):435-446.
Masala L, Ariu F, Bogliolo L, et al. 2018. Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes[J]. Molecular Reproduction and Development. doi: 10.1002/mrd.22977. [Epub ahead of print].
McGraw S, Zhang JX, Farag M, et al. 2015. Transient DNMT1 suppression reveals hidden heritable marks in the genome[J]. Nucleic Acids Research, 43(3):1485-1497.
Peat JR, Reik W. 2012. Incomplete methylation reprogramming in SCNT embryos[J]. Nature Genetics, 44(9):965-966.
Song X, Liu Z, He H, et al. 2017a. Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs[J]. Oncotarget, 8(21):34980-34991.
Song X, Li F, Jiang Z, et al. 2017b. Imprinting disorder in donor cells is detrimental to the development of cloned embryos in pigs[J]. Oncotarget, 8(42):72363-72374.
Wei, Y, Zhu, J, Huan, Y, et al. 2010. Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets[J]. Cellular Reprogramming, 12(2):213-222.
Yang X, Smith SL, Tian XC, et al. 2007. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning[J]. Nature Genetics, 39(3):295-302.
Yamanaka K, Sakatani M, Kubota K, et al.2011.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[J]. Journal of Reproduction and Development, 57(3):393-402.
Zamudio N, Bourc'his D. 2010. Transposable elements in the mammalian germline: a comfortable niche or a deadly trap? [J]. Heredity, 105(1):92-104.
