研究报告

利用CRISPR/Cas9系统建立Xist基因敲除猪模型

展开
  • 华南农业大学动物科学学院,国家生猪种业工程技术研究中心,广州 510642
李国玲,硕士研究生,专业方向:基因编辑。E-mail: 792268184@qq.com

收稿日期: 2016-04-19

  修回日期: 2016-06-18

  网络出版日期: 2016-10-09

基金资助

国家转基因重大专项(编号:2016ZX08006002)和广东省科学技术部(编号:2016B0202330064,2015TX01N081)资助

Establishment of porcine Xist knockout model using CRISPR/Cas9 system

Expand
  • National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China

Received date: 2016-04-19

  Revised date: 2016-06-18

  Online published: 2016-10-09

摘要

体细胞核移植技术在家畜良种繁育、基因修饰动物生产、濒危动物的拯救和人类疾病的治疗等领域具有重要的应用价值,但目前克隆动物生产效率较低,平均不超过5%。低下的克隆效率极大地限制了该技术的快速发展。在影响克隆猪生产效率的诸多因素中,X染色体的异常失活是导致克隆效率低下的重要原因,而与X染色体失活密切相关的一个重要基因是Xist基因,这表明Xist基因可能直接或间接地影响猪的克隆效率。本文以CRISPR/Cas系统为基础,在Xist基因上设计5个CRISPR/Cas系统打靶位点,并筛选出有效的Target 3、Target 4 sgRNA,在细胞水平切割效率为1%和3%,在胚胎水平为75%和85.7%。同时将有效的sgRNA体外转录并显微注射至胚胎体内,发现Target 3和Target 4组合效果最好,敲除效率为100%。通过胞浆注射和胚胎移植方法生产出6头克隆猪,有2头活仔实现完全敲除。本文成功建立Xist基因敲除猪模型,为后续通过敲除猪Xist基因提高克隆效率的研究奠定了基础。

关键词: Xist; CRISPR/Cas; 克隆效率;

本文引用格式

李国玲, 钟翠丽, 倪生, 刘德武, 蔡更元, 李紫聪, 杨化强, 吴珍芳 . 利用CRISPR/Cas9系统建立Xist基因敲除猪模型[J]. 遗传, 2016 , 38(12) : 1081 -1089 . DOI: 10.16288/j.yczz.16-137

Abstract

Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

参考文献

[1] Wise J. Dolly the sheep was a clone, Edinburgh scientist maintains. BMJ , 1998, 316(7131): 573.
[2] Wakayama T, Perry ACF, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature , 1998, 394(6691): 369-374.
[3] Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overström EW, Echelard Y. Production of goats by somatic cell nuclear transfer. Nat Biotechnol , 1999, 17(5): 456-461.
[4] Kitiyanant Y, Saikhun J, Chaisalee B, White KL, Pavasuthipaisit K. Somatic cell cloning in Buffalo ( Bubalus bubalis ): effects of interspecies cytoplasmic recipients and activation procedures. Cloning Stem Cells , 2001, 3(3): 97-104.
[5] Chesné P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol , 2002, 20(4): 366-369.
[6] Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A, Cozzi J. Generation of fertile cloned rats by regulating oocyte activation. Science , 2003, 302(5648): 1179.
[7] Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Shamim M, Kim JJ, Kang SK, Schatten G, Hwang WS. Dogs cloned from adult somatic cells. Nature , 2005, 436(7051): 641.
[8] Li ZY, Sun XS, Chen J, Liu XM, Wisely SM, Zhou Q, Renard JP, Leno GH, Engelhardt JF. Cloned ferrets produced by somatic cell nuclear transfer. Dev Biol , 2006, 293(2): 439-448.
[9] Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai YF, Boone J, Walker S, Ayares DL, Colman A, Campbell KHS. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature , 2000, 407(6800): 86-90.
[10] Lagutina I, Fulka H, Lazzari G, Galli C. Interspecies somatic cell nuclear transfer: advancements and problems. Cell Reprogram , 2013, 15(5): 374-384.
[11] Zhao JG, Whyte J, Prather RS. Effect of epigenetic regulation during swine embryogenesis and on cloning by nuclear transfer. Cell Tissue Res , 2010, 341(1): 13-21.
[12] Whitworth KM, Li RF, Spate LD, Wax DM, Rieke A, Whyte JJ, Manandhar G, Sutovsky M, Green JA, Sutovsky P, Prather RS. Method of oocyte activation affects cloning efficiency in pigs. Mol Reprod Dev , 2009, 76(5): 490-500.
[13] Nánássy L, Lee K, Jávor A, Macháty Z. Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos. Anim Reprod Sci , 2008, 104(2-4): 264-274.
[14] McGraw S, Oakes CC, Martel J, Cirio MC, De Zeeuw P, Mak W, Plass C, Bartolomei MS, Chaillet JR, Trasler JM. Loss of DNMT1o disrupts imprinted X chromosome inactivation and accentuates placental defects in females. PLoS Genet , 2013, 9(11): e1003873.
[15] Park SJ, Park HJ, Koo OJ, Choi WJ, Moon JH, Kwon DK, Kang JT, Kim S, Choi JY, Jang G, Lee BC. Oxamflatin improves developmental competence of porcine somatic cell nuclear transfer embryos. Cell Reprogram , 2012, 14(5): 398-406.
[16] Wutz A. Gene silencing in X-chromosome inactivation: advances in understanding facultative heterochromatin formation. Nat Rev Genet , 2011, 12(8): 542-553.
[17] Wutz A, Rasmussen TP, Jaenisch R. Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat Genet , 2002, 30(2): 167-174.
[18] Inoue K, Kohda T, Sugimoto M, Sado T, Ogonuki N, Matoba S, Shiura H, Ikeda R, Mochida K, Fujii T, Sawai K, Otte AP, Tian XC, Yang XZ, Ishino F, Abe K, Ogura A. Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer. Science , 2010, 330(6003): 496-499.
[19] Matoba S, Inoue K, Kohda T, Sugimoto M, Mizutani E, Ogonuki N, Nakamura T, Abe K, Nakano T, Ishino F, Ogura A. RNAi-mediated knockdown of Xist can rescue the impaired postimplantation developmen
文章导航

/