研究报告

转基因克隆牛胎盘中印迹基因PEG10的DNA甲基化水平

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  • 西北农林科技大学动物医学院, 农业部动物生殖生理和胚胎工程重点实验室, 杨凌 712100

收稿日期: 2010-10-14

  修回日期: 2010-12-09

  网络出版日期: 2011-05-25

基金资助

国家转基因生物新品种培育科技重大专项(编号:2008ZX08007-004)资助

The methylation status of PEG10 in placentas of cloned transgenic calves

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  • College of Veterinary Medicine, Northwest A&F University, Key Laboratory of Animal Reproductive Endocrinology & Embryo Bio-technology, Ministry of Agriculture, Yangling 712100, China

Received date: 2010-10-14

  Revised date: 2010-12-09

  Online published: 2011-05-25

摘要

低效率的体细胞核移植技术显著制约着该技术在转基因动物生产上的广泛应用。目前认为供体细胞核不能被受体卵母细胞胞质完全的表观重编程是其效率低下的最主要原因, 而DNA甲基化是基因表观修饰的主要方式之一。为了探求转基因克隆牛的死亡是否与其胎盘中印迹基因的甲基化的重编程程度相关, 文章通过亚硫酸氢盐测序法(Bisulfite sequencing PCR, BSP)和亚硫酸氢盐联合限制性内切酶分析法(Combined bisulfite restriction analysis, COBRA), 对印迹基因PEG10在围产期死亡且存在发育缺陷的转基因克隆牛的胎盘 (死亡组)和存活的转基因克隆牛的胎盘(存活组)与正常对照牛胎盘(对照组)的DNA甲基化水平进行了详细的比较。结果发现, 与对照组相比, PEG10基因在死亡组上表现出异常的超甲基化水平, 而存活组与对照组相比无显著性差异。研究结果显示, 胎盘中印迹基因的DNA甲基化表观重编程不彻底可能是导致转基因克隆牛发育异常进而死亡的主要原因之一。

本文引用格式

苏建民,许文兵,李艳艳,王丽君,王勇胜,张涌 . 转基因克隆牛胎盘中印迹基因PEG10的DNA甲基化水平[J]. 遗传, 2011 , 33(5) : 533 -538 . DOI: 10.3724/SP.J.1005.2011.00533

Abstract

The low efficiency of somatic cell nuclear transfer (SCNT) is a significant barrier to the production of highly valuable transgenic livestock. It is generally believed that the principal cause of the low SCNT efficiency is the aberrant nuclear epigenetic reprogramming of donor somatic cell. DNA methylation is a major epigenetic modification of the genome and plays a crucial role in nuclear reprogramming during SCNT. In order to assess whether the abnormal epigenetic modifications of the imprinted gene in placenta are correlated with the development abnormality and death of the cloned transgenic calves, the DNA methylation patterns of PEG10 were compared in the placentas from different kinds of cattle. This comparison included transgenic cloned calves died during perinatal stage and showed developmental defects (Death group), transgenic cloned calves survived and lived on healthily (Live group) and the normal reproduced calves (N group) used as the control group analyzed by Bisulfite Sequencing PCR (BSP) method and Combined Bisulfite Restriction Analysis (COBRA). Comparing to the control group, PEG10 gene in the Death group showed abnormal hyper-methylation, but was not significant different in methylation level from the Live group. It can be postulated from the results that the incomplete or abnormal DNA methylation epigenetic reprogramming of imprinting gene in placenta may be one of the main causes of the abnormal development and death of the transgenic cloned cattle.

参考文献

[1] Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL. Production of transgenic rabbits, sheep and pigs by microinjection. Nature, 1985, 315(6021): 680-683.
[2] Campbell KH, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature, 1996, 380(6569): 64-66.
[3] Cibelli JB, Campbell KH, Seidel GE, West MD, Lanza RP. The health profile of cloned animals. Nat Biotechnol, 2002, 20(1): 13-14.
[4] Constant F, Guillomot M, Heyman Y, Vignon X, Laigre P, Servely JL, Renard JP, Chavatte-Palmer P. Large offspring or large placenta syndrome? Morphometric analysis of late ges-tation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois. Biol Re-prod, 2006, 75(1): 122-130.
[5] Chavatte-Palmer P, Heyman Y, Richard C, Monget P, Le-Bourhis D, Kann G, Chilliard Y, Vignon X, Renard JP. Clini-cal, hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells. Biol Re-prod, 2002, 66(6): 1596-1603
[6] Miglino MA, Pereira FT, Visintin JA, Garcia JM, Meirelles FV, Rumpf R, Ambrósio CE, Papa PC, Santos TC, Carvalho AF, Leiser R, Carter AM. Placentation in cloned cattle: Structure and microvascular architecture. Theriogenology, 2007, 68(4): 604-617.
[7] Yang L, Chavatte-Palmer P, Kubota C, O'neill M, Hoagland T, Renard JP, Taneja M, Yang X, Tian XC. Expression of imprinted genes is aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones. Mol Reprod Dev, 2005, 71(4): 431-438.
[8] Inoue K, Kohda T, Lee J, Ogonuki N, Mochida K, Noguchi Y, Tanemura K, Kaneko-Ishino T, Ishino F, Ogura A. Faithful expression of imprinted genes in cloned mice. Science, 2002, 295(5553):297-297.
[9] Wei Y, Zhu J, Huan Y, Liu Z, Yang C, Zhang X, Mu Y, Xia P, Liu Z. Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets. Cell Reprogram, 2010, 12(2): 213-222.
[10] 郭磊, 李慧, 韩之明. DNA甲基化和组蛋白修饰在克隆动物发育过程中的作用. 遗传, 2010, 32(8): 762-768.
[11] 杨荣荣, 李相运. DNA甲基化与克隆动物的发育异常. 遗传, 2007, 29(9): 1043-1048.
[12] 苏建民, 华松, 张涌. 基因组印迹的调控机制及其对动物克隆的影响. 自然科学进展, 2009, 19(8): 798-805.
[13] Tsou AP, Chuang YC, Su JY, Yang CW, Liao YL, Liu WK, Chiu JH, Chou CK. Overexpression of a novel imprinted gene, PEG10, in human hepatocellular carcinoma and in re-generating mouse livers. J Biomed Sci, 2003, 10(6): 625-635.
[14] 张琼, 叶达伟, 常莹, 宋宇虎, 谢娜, 王晓燕, 林菊生. PEG10 基因在不同转移潜能肝癌细胞中表达的定量分析. 华中科技大学学报(医学版), 2006, 35(6): 758-759.
[15] Kainz B, Shehata M, Bilban M, Kienle D, Heintel D, Krömer-Holzinger E, Le T, Kröber A, Heller G, Schwarzinger I, Demirtas D, Chott A, Döhner H, Zöchbauer-Müller S, Fonatsch C, Zielinski C, Stilgenbauer S, Gaiger A, Wagner O, Jäger U. Overexpression of the paternally expressed gene 10 (PEG10) from the imprinted locus on chromosome 7q21 in high-risk B-cell chronic lymphocytic leukemia. Int J Cancer, 2007, 121(9): 1984-1993.
[16] Smallwood A, Papageorghiou A, Nicolaides K, Alley MK, Alice J, Nargund G, Ojha K, Campbell S, Banerjee S. Temporal regulation of the expression of syncytin (HERV-W), maternally imprinted PEG10, and SGCE in human p lacenta. Biol Rep Rod, 2003, 69 (1): 286-293.
[17] Liu JH, Yin S, Xiong B, Hou Y, Chen DY, Sun QY. Aberrant DNA methylation imprints in aborted bovine clones. Mol Reprod Dev, 2008, 75(4): 598-607.
[18] 彭巍, 兰志刚, 马晶晶, 王保垒, 张涌. HBD-3基因的乳腺特异性表达载体的构建及真核表达. 生物工程学报, 2009, 25(7): 968-974.
[19] 马晶晶, 王勇胜, 何小宁, 郑月茂, 张涌. 牛胎儿成纤维细胞β防御素(hBD3)基因转染及转基因克隆胚制备. 农业生物技术学报, 2010, 18(4): 707-712.
[20] Wang YS, Xiong XR, An ZX, Wang LJ, Liu J, Quan FS, Hua S, Zhang Y. Production of cloned calves by combination treatment of both donor cells and early cloned embryos with 5-aza-2/-deoxycytidine and trichostatin A. The
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