甲基化酶抑制剂5-Aza-CdR处理对德保猪手工克隆胚胎体外发育潜能的影响

doi:10.11843/j.issn.0366-6964.2014.08.010

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (8): 1274-1281.doi: 10.11843/j.issn.0366-6964.2014.08.010

甲基化酶抑制剂5-Aza-CdR处理对德保猪手工克隆胚胎体外发育潜能的影响

陆杏蓉¹，孙俊铭¹，李志鹏¹，李兰玉¹，谢炳坤²，刘庆友¹，石德顺^1*，崔奎青^1*

（1.广西大学亚热带农业生物资源保护与利用国家重点实验室，南宁 530005； 2.广西壮族自治区畜牧研究所，南宁 530005）

收稿日期:2014-01-12 出版日期:2014-08-23 发布日期:2014-08-23
通讯作者: 石德顺，E-mail: ardsshi@gxu.edu.cn；崔奎青，E-mail: kqcui@126.com
作者简介:陆杏蓉（1987-），女，广西百色人，硕士生，主要从事动物胚胎工程研究，E-mail:luxingrong074@163.com
基金资助:
国家转基因重大专项（2011ZX08007-003）；国家自然科学基金（31160457）；桂科基金（2010GXNSFA013097）

Effect of Methyltransferase Inhibitor 5-Aza-CdR on Development Potency of Handmade Cloning Embryo of Debao Pig

LU Xing-rong¹，SUN Jun-ming¹，LI Zhi-peng¹，LI Lan-yu¹，XIE Bing-kun²，LIU Qing-you¹，SHI De-shun^1*，CUI Kui-qing^1*

(1.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources，Guangxi University,Nanning 530005, China; 2.Guangxi Zhuang Autonomous Region Animal Husbandry Institute,Nanning 530005, China）

Received:2014-01-12 Online:2014-08-23 Published:2014-08-23

摘要/Abstract

摘要：

供体细胞的不完全重编程是动物克隆效率低的主要原因。本研究采用不同浓度（0.00（对照）、0.25、0.50和1.00 μmol•L^-1）的DNA甲基化转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-2′-deoxycytidine, 5-Aza-CdR)处理德保猪耳成纤维细胞，将处理后成纤维细胞作供体进行手工克隆。结果显示：各处理组细胞生长曲线均呈“S”型，其中0.25 μmol•L^-1组细胞生长曲线与对照组最相近。随着5-Aza-CdR浓度增加，细胞均有不同程度的畸变、生长抑制甚至致死。0.00~0.50 μmol•L^-1 5-Aza-CdR处理对德保猪成纤维细胞不会显著改变其细胞染色体整倍性。5-Aza-CdR不同程度下调了德保猪耳成纤维细胞中Dnmt1、Tet1、Tet2和Tet3的相对表达，对Dnmt1、Tet1和Tet3表达量的下调呈剂量相关。以处理后成纤维细胞作供体，猪手工克隆重构胚体外发育的卵裂率（24、48 h）和桑椹胚率各组间差异不显著（P>0.05），其中0.25 μmol•L^-1 组和0.50 μmol•L^-1 组囊胚发育率显著高于1.00 μmol•L^-1 组（P<0.05），而与对照组差异不显著（P>0.05）。0.50 μmol•L^-1组囊胚细胞数显著高于对照组和1.00 μmol•L^-1 组（P<0.05），而与0.25 μmol•L^-1 组之间差异不显著（P>0.05）。综上表明：高浓度5-Aza-CdR对德保猪成纤维细胞增殖和染色体具有副作用，低浓度（≤0.25 μmol•L^-1）的5-Aza-CdR可用于供体处理以降低克隆胚胎甲基化水平及提高手工克隆猪胚胎发育潜能。

Abstract:

The inefficiency of cloned animal producing is mainly due to uncompleted epigenetic reprogramming. In this study, the effect of Debao pig ear fibroblasts treated with different concentrations (0.00（control）, 0.25, 0.50 and 1.00 μmol•L^-1) of 5-Aza-CdR (DNA methyltransferase inhibitor) on the developmental potency of handmade cloned pig embryos were investigated. The results showed that all treated cells have “S” shape growth curve, meantime cells treated with 0.25 μmol•L-1 5-Aza-CdR have a growth curve which was similar to that of control group’s. With the concentrations increasing of 5-Aza-CdR, cells showed distortion, growth inhibition in a certain degree, and even death. Debao pig fibroblasts treated with 5-Aza-CdR from 0.00 to 0.50 μmol•L^-1 didn’t show significantly alteration on the chromosome ploidy.5-Aza-CdR could down regulate the expression of Dnmt1, Tet1, Tet2 and Tet3 on Debao pig ear fibroblasts following a dose-related quantity pattern. The cleavage and morula rates of handmade cloned embryos with donor cell treated with 5-Aza-CdR had no significant difference with that of the control group (P>0.05). However, the blastocyst rates of 0.25 μmol•L^-1 and 0.50 μmol•L^-1 treatment groups were significantly higher than that of 1.00 μmol•L^-1 group (P<0.05), but had no significant difference with that of the control group (P>0.05). The total blastocyst cells number of 0.50 μmol•L^-1 treatment groups was significantly higher than that of control group and 1.00 μmol•L^-1 group (P<0.05), but had no significant difference with that of 0.25 μmol•L^-1 group (P>0.05). In conclusion,high concentration of 5-Aza-CdR have side effect on the proliferation and chromosome ploidy of Debao pig fibroblasts. Applying low concentration (≤0.25 μmol•L^-1) of 5-Aza-CdR on donor cells reduced the methylation level of cloned embryos and consequently enhanced the developmental potency of handmade cloned pig embryos.

中图分类号:

陆杏蓉，孙俊铭，李志鹏，李兰玉，谢炳坤，刘庆友，石德顺，崔奎青. 甲基化酶抑制剂5-Aza-CdR处理对德保猪手工克隆胚胎体外发育潜能的影响[J]. 畜牧兽医学报, 2014, 45(8): 1274-1281.

LU Xing-rong，SUN Jun-ming，LI Zhi-peng，LI Lan-yu，XIE Bing-kun，LIU Qing-you，SHI De-shun，CUI Kui-qing. Effect of Methyltransferase Inhibitor 5-Aza-CdR on Development Potency of Handmade Cloning Embryo of Debao Pig[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1274-1281.

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甲基化酶抑制剂5-Aza-CdR处理对德保猪手工克隆胚胎体外发育潜能的影响

Effect of Methyltransferase Inhibitor 5-Aza-CdR on Development Potency of Handmade Cloning Embryo of Debao Pig

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