The Effect of DNA Methyltransferase Inhibitor 5-Aza-CdR on AID Gene-modified Bovine Fetal Fibroblasts

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.016

Abstract

Abstract: In order to improve the efficiency of somatic cell nuclear transfer,the researchers used drugs which can alter DNA methylation or histone modifications to benefit somatic cell nuclear transfer. Among them,5-aza-2'-deoxycytidine（5-Aza-CdR）blocked DNA methylation by inhibiting methyl group transfer to adenine or cytosine,and ultimately reduced genomic methylation. In this study,the AID-overexpressing and AID-knocked-down cells were used to study the effects of 5-Aza-CdR（1,2,3,4,and 5 µmol/L）on cell morphology,cell cycle,related gene expression and the changes of methylation status in the gene promoter region. Additionally,the differences between genomic DNA demethylation and site-specific DNA demethylation was analyzed,the methods and its action mechanism of increasing the reprogramming efficiency of somatic cells were discussed. Real-time PCR,BSP（Bisulfite Sequencing PCR）,Western blotting,and flow cytometry were employed to analyze the effects of 5-Aza-CdR on the AID-transgenic and AID-knocked-down cells. The results showed that 5-Aza-CdR presented dose-dependent effect on AID-transgenic cells,while the 5-Aza-CdR concentration was 4 µmol/L,obvious toxicity was observed as a lot of cells were dead（P<0.05）. When treated by 1-3 µmol/L,the cell’s morphology changed,and proliferation of cells was inhibited. Karyotype analysis showed that 3 µmol/L treatments resulted in the emergence of a small proportion of aneuploidy cells,indicating that high concentrations of 5-Aza-CdR increased the rate of aneuploidy. The number of cells which expressed red fluorescent protein（DsRed）significantly increased after 1 µmol/L 5-Aza-CdR treatment,compared to the control group,the expression of AID and SOX2 were also increased,and the methylation levels in SOX2 promoter region were somehow decreased. After treated by 5-Aza-CdR,the expression of the OCT4 and SOX2genes increased in AID-knocked-down cells,but the expression of the NANOG did not change. The above results revealed that 5-Aza-CdR treatment affected the cell morphology,cell cycle and reporter gene expression in bovine transgenic cells. In conclusion,while treated by 5-Aza-CdR,the genomic demethylation and loci-specific demethylation of AID act synergistically in somatic cell reprogramming.

Key words: 5-Aza-CdR, AID, DNA demethylation, somatic cell nuclear transfer

AO Xu-dong ,SA Ru-la, WANG Jie, WANG Hui-min ,YU Hai-quan. The Effect of DNA Methyltransferase Inhibitor 5-Aza-CdR on AID Gene-modified Bovine Fetal Fibroblasts[J]. Biotechnology Bulletin, 2016, 32(8): 103-112.

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