锌指核酸酶技术制备肌肉生长抑制素基因敲除的五指山小型猪成纤维细胞

doi:10.3724/SP.J.1005.2013.00778

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HEREDITAS ›› 2013, Vol. 35 ›› Issue (6): 778-785.doi: 10.3724/SP.J.1005.2013.00778

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Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases

CAO Sui-Zhong¹, YUE Cheng-He^{1, 2}, LI Xi-Rui², FENG Chong², LONG Chuan², PAN Deng-Ke²

1. College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2012-11-01 Revised:2012-12-07 Online:2013-06-20 Published:2013-06-25

Abstract

Abstract: Disruption of myostatin (MSTN) gene in pigs may improve porcine lean meat percentage (LMP), and create an animal model for certain human diseases. Using zinc-finger nucleases (ZFNs) technology, MSTN gene was deleted in Wuzhishan miniature pig fibroblasts by transfection of either ZFNs plasmids or ZFNs mRNA in high efficiency. Strikingly, ZFNs encoding mRNA could produce MSTN+/-and MSTN-/- cell colonies with single or double allele deletion by single transfection. Sequencing results demonstrated that 92.18% of the mutations were short fragment deletions or insertions (≤10 bp). Prediction of amino acids sequences indicated that more than half of the mutations cause premature transla-tional-termination codon. MSTN+/+, MSTN+/-, and MSTN-/- cell colonies were used as nuclear donor for somatic cell nuclear transfer (SCNT), and developmental potential of SCNT embryos were measured by the blastocyst rate. The results revealed no significant difference in development competence among the three kinds of reconstructed embryos (14.29% vs. 19.64% vs. 16.13%), which provides the possibility of making myostatin knock out pigs in the future.

Key words: zinc-finger nucleases (ZFNs), myostatin, gene knockout, Wuzhishan miniature pigs (WZSP), MSTN-/- fibroblasts

CAO Sui-Zhong YUE Cheng-He LI Xi-Rui FENG Chong LONG Chuan PAN Deng-Ke. Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases[J]. HEREDITAS, 2013, 35(6): 778-785.

References

[1] 许振英. 中国地方猪种种质特性. 杭州: 浙江科学技术出版社, 1989.
[2] Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Ménissier F, Massa-banda J, Fries R, Hanset R, Georges M. A deletion in the bovine myostatin gene causes the double-muscled pheno-type in cattle. Nat Genet, 1997, 17(1): 71-74.
[3] McFarlane C, Hui GZ, Amanda WZW, Lau HY, Lokireddy S, Ge XJ, Mouly V, Butler-Browne G, Gluckman PD, Sharma M, Kambadur R. Human myostatin negatively regulates human myoblast growth and differentiation. Am J Physiol Cell Physiol, 2011, 301(1): C195-C203.
[4] McPherron AC, Lawler AM, Lee SJ. Regulation of skele-tal muscle mass in mice by a new TGF-p superfamily member. Nature, 1997, 387(6628): 83-90.
[5] McPherron AC, Lee SJ. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA, 1997, 94(23): 12457-12461.
[6] Stinckens A, Luyten T, Bijttebier J, Van den Maagdenberg K, Dieltiens D, Janssens S, De Smet S, Georges M, Buys N. Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscular-ity. Anim Genet, 2008, 39(6): 586-596.
[7] Mendias CL, Bakhurin KI, Faulkner JA. Tendons of my-ostatin-deficient mice are small, brittle, and hypocellular. Proc Natl Acad Sci USA, 2008, 105(1): 388-393.
[8] Wilkes JJ, Lloyd DJ, Gekakis N. Loss-of-function muta-tion in myostatin reduces tumor necrosis factor α produc-tion and protects liver against obesity-induced insulin re-sistance. Diabetes, 2009, 58(5): 1133-1143.
[9] Bibikova M, Beumer K, Trautman JK, Carroll D. Enhanc-ing gene targeting with designed zinc finger nucleases. Science, 2003, 300(5620): 764.
[10] Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui XX, Meng XD, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R. Knockout rats via embryo microin-jection of zinc-finger nucleases. Science, 2009, 325(5939): 433.
[11] Hauschild J, Petersen B, Santiago Y, Queisser AL, Carn-wath JW, Lucas-Hahn A, Zhang L, Meng XD, Gregory PD, Schwinzer R, Cost GJ, Niemann H. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci USA, 2011, 108(29): 12013-12017.
[12] 罗学明, 肖炜, 冯冲, 龙川, 闫军, 薛振华, 云鹏, 潘登科. 初次卵裂时间是猪克隆胚胎发育潜能的重要标识. 生物化学与生物物理进展, 2010, 37(12): 1339-1345.
[13] Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem, 2000, 275(51): 40235-40243.
[14] Dai YF, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL. Targeted disruption of the α1, 3-galactosyltransferase gene in cloned pigs. Nat Biotechnol, 2002, 20(3): 251-255.
[15] Kolber-Simonds D, Lai LX, Watt SR, Denaro M, Arn S, Augenstein ML, Betthauser J, Carter DB, Greenstein JL, Hao YH, Im GS, Liu ZH, Mell GD, Murphy CN, Park KW, Rieke A, Ryan DJ, Sachs DH, Forsberg EJ, Prather RS, Hawley RJ. Production of α-1, 3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci USA, 2004, 101(19): 7335-7340.
[16] Lai LX, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS. Production of α-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 2002, 295(5557): 1089-1092.
[17] Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai YF, Ayares DL. Production of α1, 3-galactosyltransfe

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Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases

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