Xist基因抑制对牛SCNT早期胚胎发育的影响

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.033

生物技术通报 ›› 2015, Vol. 31 ›› Issue (1): 214-219.doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.033

Xist基因抑制对牛SCNT早期胚胎发育的影响

刘黎明, 孙伟², 张金吨, 赵丽霞², 李树裕² , 王标, 陈杨林, 胡树香², 吴宝江², 李喜和^1，2

（1.内蒙古大学蒙古高原动物遗传资源研究中心，呼和浩特010021；2.内蒙古赛科星繁育生物技术股份有限公司，呼和浩特011517）

收稿日期:2014-05-27 出版日期:2015-01-09 发布日期:2015-01-10
作者简介:刘黎明，男，硕士，研究方向：动物生殖生物学与生物技术；E-mail：nmgdxllm@163.com
基金资助:
内蒙古自治区家畜性控精液技术熟化与产业化开发（20111701）

Effect of Xist Gene Repression on Early Development of Bovine SCNT Embryos

Liu Liming, Sun Wei², Zhang Jindun, Zhao Lixia², Li Shuyu², Wang Biao, Chen Yanglin, Hu Shuxiang², Wu Baojiang², Li Xihe^1，2

（1.Research Center for Animal Genetic Resources of Mongolia Plateau，Inner Mongolia University，Hohhot 010021；2. Inner Mongolia Saikexing Reproductive Biotechnology Co.，Ltd.，Hohhot 011517）

Received:2014-05-27 Published:2015-01-09 Online:2015-01-10

摘要/Abstract

摘要： Xist是与X染色体失活相关的非编码基因，它在合子期基因组开始表达，是胚胎发育早期表达的第一个印记基因。探讨了特异性抑制Xist的TALER-REPRESSOR（TALER）载体转染到胎牛成纤维细胞对Xist基因的抑制作用，并以抑制Xist基因表达的细胞作为核供体制作克隆胚胎，研究Xist基因抑制对牛克隆胚早期发育的影响。结果显示，与对照组细胞相比，TALER载体将Xist相对表达量下调了93.85%，说明本试验设计的载体转染系统能够有效抑制Xist基因的表达。选取Xist抑制表达阳性的转染细胞用于体细胞核移植试验，克隆胚胎发育结果显示，试验组和对照组的卵裂率、8细胞发育率、桑葚胚发育率和囊胚发育率分别为78.8% vs 75.1%（P>0.05，无显著差异）、54.4% vs 50.6%（P>0.05，无显著差异）、12.3% vs 27.8%（P<0.01，差异极显著）、0 vs 26.6%（P<0.01，差异极显著）。综上所述，试实验设计的特异性抑制Xist表达的TALER载体可有效抑制雌性胎牛成纤维细胞中Xist的表达。供体细胞Xist这种基因下调可使克隆胚胎2-8细胞率略有提升，但囊胚期和桑葚胚率明显降低。因此，其机制尚待于进一步探讨。

关键词: Xist基因, TALER载体, 胎牛成纤维细胞, 克隆胚胎早期发育

Abstract: Xist is an X-inactivation related gene that produces a noncoding RNA, and it is one of the first imprinted genes to be expressed in the early embryo with expression beginning at zygotic genome activation. This experiment aimed at investigating Xist repression in fetal bovine fibroblast by TALE-REPRESSOR（TALER）which contains mCherry as reporter, and the impact of Xist repression on early development of cloned cattle embryos using the transfected cells as nuclei donor. The results showed TALER repressed Xist gene expression by 93.85% in fluorescence-activated sorted cells compared with wild type fibroblast, indicating the TALER vector designed in this experiment effectively suppressed expression of Xist. The mCherry positive cells were selected for somatic cell nuclear transfer（SCNT）. The 2-cell, 8-cell, morula and blastocyst rates of suppressed group vs. control group were 78.8% vs 75.1%（P>0.05）, 54.4% vs. 50.6%（P>0.05）, 12.3% vs. 27.8%（P<0.01）and 0 vs. 26.6%（P<0.01）, respectively. These results indicated that TALER vectors effectively inhibited Xist gene expression in female fetal bovine fibroblast. Suppression of Xist gene promotes the 2-8 cell embryonic development of cloned embryo, however, development of morula/blastocyst decreased significantly. Therefore, the mechanism by which Xist gene expression regulates early embryonic development of cloned embryos needs further investigation.

Key words: Xist gene, TALER vector, fetal bovine fibroblast, early embryonic development

刘黎明,,孙伟,,张金吨,,赵丽霞,,李树裕,,,王标,,陈杨林,,胡树香,,吴宝江,,李喜和. Xist基因抑制对牛SCNT早期胚胎发育的影响[J]. 生物技术通报, 2015, 31(1): 214-219.

Liu Liming, Sun Wei, Zhang Jindun, Zhao Lixia, Li Shuyu, Wang Biao, Chen Yanglin, Hu Shuxiang, Wu Baojiang, Li Xihe. Effect of Xist Gene Repression on Early Development of Bovine SCNT Embryos[J]. Biotechnology Bulletin, 2015, 31(1): 214-219.

参考文献

[1] Zuccotti M, Boiani M, Ponce R, et al. Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock[J]. Molecular Reproduction and Development, 2002, 61（1）：14-20.
[2] Payer B, Lee JT. X chromosome dosage compensation：how mam-mals keep the balance[J]. Annual Review of Genetics, 2008, 42：733-772.
[3] Fukuda A, Cao F, Morita S, et al. Identification of inappropriately reprogrammed genes by large-scale transcriptome analysis of individual cloned mouse blastocysts[J]. PLoS One, 2010, 5（6）：e11274.
[4] Nolen LD, Gao S, Han Z, et al. X chromosome reactivation and regulation in cloned embryos[J]. Developmental Biology, 2005, 279（2）：525-540.
[5] Inoue K, Kohda T, Sugimoto M, et al. Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer[J]. Science, 2010, 330（6003）：496-499.
[6] Mahfouz MM, Li L, Piatek M, et al. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein[J]. Plant Molecular Biology, 2012, 78（3）：311-321.
[7] Garg A, Lohmueller JJ, Silver PA, et al. Engineering synthetic TAL effectors with orthogonal target sites[J]. Nucleic Acids Research, 2012, 40（15）：7584-7595.
[8] Konermann S, Brigham MD, Trevino AE, et al. Optical control of mammalian endogenous transcription and epigenetic states[J]. Nature, 2013, 500（7463）：472-476.
[9] Iyengar S, Farnham PJ. KAP1 protein：an enigmatic master regulator of the genome[J]. Journal of Biological Chemistry, 2011, 286（30）：26267-26276.
[10] O’Geen H, Squazzo S L, Iyengar S, et al. Genome-wide analysis of KAP1 binding suggests autoregulation of KRAB-ZNFs[J]. PLoS Genetics, 2007, 3（6）：e89.
[11] Mahfouz MM, Li L, Shamimuzzaman M, et al. De novo-engineered transcription activator-like effector（TALE）hybrid nuclease with novel DNA binding specificity creates double-strand breaks[J]. Proceedings of the National Academy of Sciences, 2011, 108（6）：2623-2628.
[12] Cong L, Zhou R, Kuo Y, et al. Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains[J]. Nature Communications, 2012, 3：968.
[13] Matoba S, Inoue K, Kohda T, et al. RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos[J]. Proceedings of the National Academy of Sciences, 2011, 108（51）：20621-20626.
[14] 鲁成龙. pIKX-EGFP-C1 载体的构建及其对克隆胚 X 相关基因的影响[D] . 杨凌：西北农林科技大学, 2012.
[15] Dean W, Santos F, Stojkovic M, et al. Conservation of methylation reprogramming in mammalian development：aberrant reprogramming in cloned embryos[J]. Proceedings of the National Academy of Sciences, 2001, 98（24）：13734-13738.
[16] Ferreira AR, Machado GM, Diesel TO, et al. Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos[J]. Molecular Reproduction and Development, 2010, 77（7）：615-621.
[17] Ogura A, Inoue K, Ogonuki N, et al. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells[J]. Biology of Reproduction, 2000, 62（6）：1579-1584.
[18] Zakhartchenko V, Mueller S, Alberio R, et al. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts[J]. Molecular Reproduction and Development, 2001, 60（3）：362-369.

Xist基因抑制对牛SCNT早期胚胎发育的影响

Effect of Xist Gene Repression on Early Development of Bovine SCNT Embryos

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