利用CRISPR/Cas9系统建立Xist基因敲除猪模型

doi:10.16288/j.yczz.16-137

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Hereditas(Beijing) ›› 2016, Vol. 38 ›› Issue (12): 1081-1089.doi: 10.16288/j.yczz.16-137

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Establishment of porcine Xist knockout model using CRISPR/Cas9 system

Guoling Li, Cuili Zhong, Sheng Ni, Dewu Liu, Gengyuan Cai, Zicong Li, Huaqiang Yang, Zhenfang Wu

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

Received:2016-04-19 Revised:2016-06-18 Online:2016-12-20 Published:2016-10-09

Abstract

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.

Key words: Xist, CRISPR/Cas, cloning efficiency, porcine

Guoling Li, Cuili Zhong, Sheng Ni, Dewu Liu, Gengyuan Cai, Zicong Li, Huaqiang Yang, Zhenfang Wu. Establishment of porcine Xist knockout model using CRISPR/Cas9 system[J]. Hereditas(Beijing), 2016, 38(12): 1081-1089.

References

[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

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Establishment of porcine Xist knockout model using CRISPR/Cas9 system

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