title: Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.
abstract: Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.
authors: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Chinese Academy of Sciences, Guangzhou, China.
authors: Wei
authors: W
authors: Deng
authors: Dongshan
authors: D
authors: Yang
authors: Bentian
authors: B
authors: Zhao
authors: Zhen
authors: Z
authors: Ouyang
authors: Jun
authors: J
authors: Song
authors: Nana
authors: N
authors: Fan
authors: Zhaoming
authors: Z
authors: Liu
authors: Yu
authors: Y
authors: Zhao
authors: Qinghong
authors: Q
authors: Wu
authors: Bayaer
authors: B
authors: Nashun
authors: Jiangjing
authors: J
authors: Tang
authors: Zhenfang
authors: Z
authors: Wu
authors: Weiwang
authors: W
authors: Gu
authors: Liangxue
authors: L
authors: Lai
references: Curr Opin Organ Transplant. 2009 Apr;14(2):148-53
references: 19469029
references: Gene Ther. 2001 Jun;8(11):864-73
references: 11423934
references: Theriogenology. 2003 Jan 1;59(1):115-23
references: 12499023
references: Int J Clin Exp Med. 2010 May 15;3(2):129-43
references: 20607039
references: Science. 2002 Feb 8;295(5557):1089-92
references: 11778012
references: Dev Comp Immunol. 2007;31(12):1211-9
references: 17499850
references: Gene Ther. 2001 May;8(10):811-7
references: 11420646
references: Reprod Biol Endocrinol. 2003 Nov 07;1:82
references: 14613542
references: Mol Cell Biol. 2008 Jul;28(13):4227-39
references: 18458056
references: Anim Reprod Sci. 2003 Dec 15;79(3-4):291-317
references: 14643109
references: Stem Cells. 2009 Mar;27(3):543-9
references: 19096035
references: Mol Reprod Dev. 2008 Jan;75(1):26-32
references: 17546633
references: Nat Biotechnol. 2004 May;22(5):589-94
references: 15064769
references: Blood. 2003 Mar 1;101(5):1759-68
references: 12406897
references: World J Gastroenterol. 2005 May 21;11(19):2885-91
references: 15902723
references: Transplantation. 1997 Jan 15;63(1):149-55
references: 9000677
references: Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):157-62
references: 19109433
references: Traffic. 2004 Aug;5(8):616-26
references: 15260831
references: Hum Gene Ther. 2003 Mar 20;14(5):415-24
references: 12691607
references: J Med Dent Sci. 2005 Jun;52(2):129-34
references: 16189885
references: Nat Biotechnol. 2006 Apr;24(4):435-6
references: 16565727
references: Nat Methods. 2009 May;6(5):363-9
references: 19337237
references: Nat Biotechnol. 2005 May;23(5):584-90
references: 15834403
references: Mol Ther. 2000 Apr;1(4):376-82
references: 10933956
references: Mol Carcinog. 2007 Aug;46(8):654-9
references: 17440928
references: J Immunol. 2001 Dec 15;167(12):7102-10
references: 11739532
references: Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):160-4
references: 7816809
references: Xenotransplantation. 2002 May;9(3):183-90
references: 11983016
references: Virol J. 2006 Mar 15;3:14
references: 16539700
references: Mol Ther. 2004 Nov;10(5):862-73
references: 15509504
references: BMC Cell Biol. 2008 Jan 11;9:2
references: 18190688
references: Science. 2008 Sep 26;321(5897):1837-41
references: 18818360
references: Science. 2008 Nov 7;322(5903):949-53
references: 18845712
references: Mol Reprod Dev. 2005 Sep;72(1):68-76
references: 15906394
references: Hum Gene Ther. 1998 Feb 10;9(3):287-93
references: 9508046
references: Mol Reprod Dev. 2010 Mar;77(3):209-21
references: 19998476
references: Xenotransplantation. 1999 Aug;6(3):194-200
references: 10503786
references: Hum Gene Ther. 2010 Nov;21(11):1631-7
references: 20528679
references: Nature. 2009 Apr 9;458(7239):771-5
references: 19252477
references: Xenotransplantation. 2005 Mar;12(2):142-8
references: 15693845
references: Transplantation. 2004 May 15;77(9):1468-71
references: 15167611