Molecular Biology Reports

, Volume 41, Issue 1, pp 209–215 | Cite as

Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases

  • Cunfang Zhang
  • Ling Wang
  • Gang Ren
  • Zhanwei Li
  • Chonghua Ren
  • Tingting Zhang
  • Kun Xu
  • Zhiying Zhang


Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probability of identifying ZFN-mediated positive targeted disruption events is small. In this paper, we used the context-dependent assembly method to synthesize two pairs of ZFNs targeted to the sheep MSTN gene. We verified the activity of these ZFNs using an mRFP-MBS-eGFP dual-fluorescence reporter system in HEK293T cells and, according to the expression level of eGFP, we obtained a pair of ZFNs that can recognize and cut the targeted MSTN site in the reporter vector. The activity of ZFN was increased by cold stimulation at 30 °C and by mutant the wildtype FokI in ZFN with its counterpart Sharkeys. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two MSTN mutant cell lines, identified by flow cytometry and sequencing, were obtained.


Gene knockout MSTN Sheep ZFN 



This study was supported by a grant from the genetically modified organisms breeding major projects of China (2013XZ08008-003).

Supplementary material

11033_2013_2853_MOESM1_ESM.doc (16 mb)
Supplementary material 1 (DOC 16387 kb)


  1. 1.
    Thomas KR, Folger KR, Capecchi MR (1986) High frequency targeting of genes to specific sites in the mammalian genome. Cell 44(3):419–428PubMedCrossRefGoogle Scholar
  2. 2.
    Robertson EJ (1991) Using embryonic stem cells to introduce mutations into the mouse germ line. Biol Reprod 44(2):238–245PubMedCrossRefGoogle Scholar
  3. 3.
    Rouet P, Smih F, Jasin M (1994) Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci 91(13):6064–6068PubMedCrossRefGoogle Scholar
  4. 4.
    Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161(3):1169–1175PubMedGoogle Scholar
  5. 5.
    Lloyd A, Plaisier CL, Carroll D, Drews GN (2005) Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci USA 102(6):2232–2237PubMedCrossRefGoogle Scholar
  6. 6.
    Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26(6):695–701PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26(6):702–708PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Meyer M, de Angelis MH, Wurst W, Kühn R (2010) Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci 107(34):15022–15026PubMedCrossRefGoogle Scholar
  9. 9.
    Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, Carroll D, Tamura T, Zurovec M (2010) Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol 40(10):759–765PubMedCrossRefGoogle Scholar
  10. 10.
    Young JJ, Cherone JM, Doyon Y, Ankoudinova I, Faraji FM, Lee AH, Ngo C, Guschin DY, Paschon DE, Miller JC (2011) Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc Natl Acad Sci 108(17):7052–7057PubMedCrossRefGoogle Scholar
  11. 11.
    Chu X, Zhang Z, Yabut J, Horwitz S, Levorse J, XQ Li, Zhu L, Lederman H, Ortiga R, Strauss J (2012) Characterization of multidrug resistance 1a/P-glycoprotein knockout rats generated by zinc finger nucleases. Mol Pharmacol 81(2):220–227PubMedCrossRefGoogle Scholar
  12. 12.
    Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Gregory PD, Urnov FD, Holmes MC (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci 104(9):3055–3060PubMedCrossRefGoogle Scholar
  13. 13.
    Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R (2011) Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci 108(29):12013–12017PubMedCrossRefGoogle Scholar
  14. 14.
    Flisikowska T, Thorey IS, Offner S, Ros F, Lifke V, Zeitler B, Rottmann O, Vincent A, Zhang L, Jenkins S (2011) Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases. PLoS ONE 6(6):e21045PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Yu S, Luo J, Song Z, Ding F, Dai Y, Li N (2011) Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Res 21(11):1638–1640PubMedCrossRefGoogle Scholar
  16. 16.
    Dong Z, Ge J, Li K, Xu Z, Liang D, Li J, Li J, Jia W, Li Y, Dong X, Cao S, Wang X, Pan J, Zhao Q (2011) Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc finger nucleases. PLoS ONE 6(12):e28897PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387(6628):83–90PubMedCrossRefGoogle Scholar
  18. 18.
    Stinckens A, Georges M, Buys N (2011) Mutations in the Myostatin gene leading to hypermuscularity in mammals: indications for a similar mechanism in fish? Anim Genet 42(3):229–234PubMedCrossRefGoogle Scholar
  19. 19.
    Chelh I, Picard B, Hocquette J, Cassar-Malek I (2011) Myostatin inactivation induces a similar muscle molecular signature in double-muscled cattle as in mice. Animal 5(02):278–286PubMedCrossRefGoogle Scholar
  20. 20.
    Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y (2010) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8(1):67–69PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Guo J, Gaj T, Barbas CF (2010) Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J Mol Biol 400(1):96–107PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Hu LY, Cui CC, Song YJ, Wang XG, Jin YP, Wang AH, Zhang Y (2012) An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA. Biotechnol Lett 34(7):1251–1255PubMedCrossRefGoogle Scholar
  23. 23.
    Van den Hoff M, Moorman A, Lamers WH (1992) Electroporation in ‘intracellular’ buffer increases cell survival. Nucleic Acids Res 20(11):2902PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE (2002) Targeted disruption of the alpha1, 3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 20(3):251–255PubMedCrossRefGoogle Scholar
  25. 25.
    Kim H, Um E, Cho SR, Jung C, Kim H, Kim JS (2011) Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 8(11):941–943PubMedCrossRefGoogle Scholar
  26. 26.
    McPherron AC, Lee SJ (1997) Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci 94(23):12457–12461PubMedCrossRefGoogle Scholar
  27. 27.
    Liu Y, Hua S, Lan J, Song Y, He Y, Quan F, Zhang Y (2010) Site-directed mutagenesis of MSTN gene by gene targeting in Qinchuan cattle. Sheng wu gong cheng xue bao 26(3):410–416 Chinese Journal of BiotechnologyPubMedGoogle Scholar
  28. 28.
    Zheng YL, Ma HM, Zheng YM, Wang YS, Zhang BW, He XY, He XN, Liu J, Zhang Y (2012) Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro. Res Vet Sci 93(2):763–769PubMedCrossRefGoogle Scholar
  29. 29.
    Kim JS, Lee HJ, Carroll D (2010) Genome editing with modularly assembled zinc-finger nucleases. Nat Methods 7(2):91PubMedCrossRefGoogle Scholar
  30. 30.
    Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31(2):294–301PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Perez-Pinera P, Ousterout DG, Brown MT, Gersbach CA (2012) Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases. Nucleic Acids Res 40(8):3741–3752PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Perez C, Guyot V, Cabaniols JP, Gouble A, Micheaux B, Smith J, Leduc S, Paques F, Duchateau P (2005) Factors affecting double-strand break-induced homologous recombination in mammalian cells. Biotechniques 39(1):109–115PubMedCrossRefGoogle Scholar
  33. 33.
    Doyon Y, Choi VM, Xia DF, Vo TD, Gregory PD, Holmes MC (2010) Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nat Methods 7(6):459–460PubMedCrossRefGoogle Scholar
  34. 34.
    Davies B, Davies G, Preece C, Puliyadi R, Szumska D, Bhattacharya S (2013) Site specific mutation of the Zic2 locus by microinjection of TALEN mRNA in mouse CD1, C3H and C57BL/6J oocytes. PLoS ONE 8(3):e60216PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Liang F, Han M, Romanienko PJ, Jasin M (1998) Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci 95(9):5172–5177PubMedCrossRefGoogle Scholar
  36. 36.
    Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435(7042):646–651PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Cunfang Zhang
    • 1
  • Ling Wang
    • 1
  • Gang Ren
    • 1
  • Zhanwei Li
    • 1
  • Chonghua Ren
    • 1
  • Tingting Zhang
    • 1
  • Kun Xu
    • 1
  • Zhiying Zhang
    • 1
  1. 1.College of Animal Science and TechnologyNorthwest A&F UniversityXianyangPeople’s Republic of China

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