The domesticated pig has emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. However, germ line-competent pluripotent porcine stem cells have not yet been derived. This has been a major obstacle to genetic modification of pigs. The transcription factor Oct4 is essential for the maintenance of pluripotency and for reprogramming somatic cells to a pluripotent state. Here, we report the production of transgenic pigs carrying an 18 kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct) to allow identification of pluripotent cells by monitoring Oct4 expression by EGFP fluorescence. Eleven viable transgenic piglets were produced by somatic cell nuclear transfer. Expression of the EGFP reporter construct was confined to germ line cells, the inner cell mass and trophectoderm of blastocysts, and testicular germ cells. Reprogramming of fibroblasts from these animals by fusion with pluripotent murine embryonic stem cells or viral transduction with human OCT4, SOX2, KLF4, and c-MYC cDNAs resulted in Oct4-EGFP reactivation. The OG2 pigs have thus proved useful for monitoring reprogramming and the induction and maintenance of pluripotency in porcine cells. In conclusion, the OG2 transgenic pigs are a new large animal model for studying the derivation and maintenance of pluripotent cells, and will be valuable for the development of cell therapy.
Get full access to this article
View all access options for this article.
References
1.
GerrardL, ZhaoD, ClarkAJ, WCui. 2005. Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency. Stem Cells, 23:124–133.
TakahashiK, SYamanaka. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.
4.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK, SYamanaka. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
5.
KuesWA, HNiemann. 2004. The contribution of farm animals to human health. Trends Biotechnol, 22:286–294.
6.
PetersenB, CarnwathJW, HNiemann. 2009. The perspectives for porcine-to-human xenografts. Comp Immunol Microbiol Infect Dis, 32:91–105.
7.
ChenLR, ShiueYL, BertoliniL, MedranoJF, BonDurantRH, GBAnderson. 1999. Establishment of pluripotent cell lines from porcine preimplantation embryos. Theriogenology, 52:195–212.
HallV. 2008. Porcine embryonic stem cells: a possible source for cell replacement therapy. Stem Cell Rev, 4:275–282.
12.
KirchhofN, CarnwathJW, LemmeE, AnastassiadisK, SchölerH, HNiemann. 2000. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol Reprod, 63:1698–1705.
13.
WuenschA, HabermannFA, KurosakaS, KloseR, ZakhartchenkoV, ReichenbachHD, SinowatzF, McLaughlinKJ, EWolf. 2007. Quantitative monitoring of pluripotency gene activation after somatic cloning in cattle. Biol Reprod, 76:983–991.
14.
YeomYI, FuhrmannG, OvittCE, BrehmA, OhboK, GrossM, HubnerK, HRSchöler. 1996. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development, 122:881–894.
15.
YoshimizuT, SugiyamaN, De FeliceM, YeomYI, OhboK, MasukoK, ObinataM, AbeK, SchölerHR, YMatsui. 1999. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ, 41:675–684.
16.
OkamotoK, OkazawaH, OkudaA, SakaiM, MuramatsuM, HHamada. 1990. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell, 60:461–472.
17.
OvittCE, HRSchöler. 1998. The molecular biology of Oct-4 in the early mouse embryo. Mol Hum Reprod, 4:1021–1031.
18.
ChengL, SungMT, Cossu-RoccaP, JonesTD, MacLennanGT, De JongJ, Lopez-BeltranA, MontironiR, LHLooijenga. 2007. OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. J Pathol, 211:1–9.
19.
PesceM, HRSchöler. 2000. Oct-4: control of totipotency and germline determination. Mol Reprod Dev, 55:452–457.
20.
KuesWA, PetersenB, MysegadesW, CarnwathJW, HNiemann. 2005. Isolation of murine and porcine fetal stem cells from somatic tissue. Biol Reprod, 72:1020–1028.
21.
NicholsJ, ZevnikB, AnastassiadisK, NiwaH, Klewe-NebeniusD, ChambersI, SchölerH, ASmith. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95:379–391.
LiW, WeiW, ZhuS, ZhuJ, ShiY, LinT, HaoE, HayekA, DengH, SDing. 2009. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell, 4:16–19.
EzashiT, TeluguBP, AlexenkoAP, SachdevS, SinhaS, RMRoberts. 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A, 106:10993–10998.
31.
WuZ, ChenJ, RenJ, BaoL, LiaoJ, CuiC, RaoL, LiH, GuY, DaiH, ZhuH, TengX, ChengL, LXiao. 2009. Generation of pig-induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol, 1:46–54.
HanDW, DoJT, GentileL, StehlingM, LeeHT, HRSchöler. 2008. Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle. Stem Cells, 26:445–454.
35.
KuesWA, CarnwathJW, PaulD, HNiemann. 2002. Cell cycle synchronization of porcine fetal fibroblasts by serum deprivation initiates a nonconventional form of apoptosis. Cloning Stem Cells, 4:231–243.
36.
HölkerM, PetersenB, HasselP, KuesWA, LemmeE, Lucas-HahnA, HNiemann. 2005. Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning Stem Cells, 7:35–44.
37.
PetersenB, Lucas-HahnA, OropezaM, HornenN, LemmeE, HasselP, QueisserAL, HNiemann. 2008. Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts. Cloning Stem Cells, 10:355–362.
38.
ChomczynskiP. 1993. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques, 15:532–534–536–537.
39.
IslamMQ, RingeJ, ReichmannE, MigottiR, SittingerM, da S MeirellesL, NardiNB, MagnussonP, KIslam. 2006. Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line. Cell Tissue Res, 326:123–137.
40.
OestrupO, HallV, PetkovSG, WolfXA, HyldigS, PHyttel. 2009. From zygote to implantation: morphological and molecular dynamics during embryo development in the pig. Reprod Domest Anim, 44,Suppl. 3:39–49.
41.
Nowak-ImialekM, KuesWA, RudolphC, SchlegelbergerB, TaylorU, CarnwathJW, HNiemann. 2010. Preferential loss of porcine chromosomes in reprogrammed interspecies cell hybrids. Cell Reprogramming, 12:55–65.
42.
BuehrM, NicholsJ, StenhouseF, MountfordP, GreenhalghCJ, KantachuvesiriS, BrookerG, MullinsJ, AGSmith. 2003. Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines. Biol Reprod, 68:222–229.
43.
GhoshZ, WilsonKD, WuY, HuS, QuertermousT, JCWu. 2010. Induced pluripotent stem cells contribute to differences with human embryonic stem cells. PLoS ONE, 5:e8975.
MiyoshiK, MoriH, MizobeY, AkasakaE, OzawaA, YoshidaM, MSato. 2009. Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos. J Reprod Dev, 55:661–669.
46.
van EijkMJ, van RooijenMA, ModinaS, ScesiL, FolkersG, van TolHT, BeversMM, FisherSR, LewinHA, RakacolliD, GalliC, de VaureixC, TrounsonAO, MummeryCL, FGandolfi. 1999. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol Reprod, 60:1093–1103.
47.
KurosakaS, EckardtS, KJMcLaughlin. 2004. Pluripotent lineage definition in bovine embryos by Oct4 transcript localization. Biol Reprod, 71:1578–1582.
48.
De FeliciM, FariniD, SDolci. 2009. In or out stemness: comparing growth factor signalling in mouse embryonic stem cells and primordial germ cells. Curr Stem Cell Res Ther, 4:87–97.
49.
De RooijDG, LDRussell. 2000. All you wanted to know about spermatogonia but were afraid to ask. J Androl, 21:776–798.
50.
WesternP. 2009. Foetal germ cells: striking the balance between pluripotency and differentiation. Int J Dev Biol, 53:393–409.
51.
KimJB, SebastianoV, WuG, Arauzo-BravoMJ, SasseP, GentileL, KoK, RuauD, EhrichM, van den BoomD, MeyerJ, HubnerK, BernemannC, OrtmeierC, ZenkeM, FleischmannBK, ZaehresH, HRSchöler. 2009. Oct4-induced pluripotency in adult neural stem cells. Cell, 136:411–419.
52.
KimJB, ZaehresH, WuG, GentileL, KoK, SebastianoV, Arauzo-BravoMJ, RuauD, HanDW, ZenkeM, HRSchöler. 2009. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature, 454:646–650.
53.
DoJT, HanDW, GentileL, Sobek-KlockeI, StehlingM, LeeHT, HRSchöler. 2007. Erasure of cellular memory by fusion with pluripotent cells. Stem Cells, 25:1013–1020.
54.
TadaM, TakahamaY, AbeK, NakatsujiN, TTada. 2001. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol, 11:1553–1558.
55.
SilvaJ, NicholsJ, TheunissenTW, GuoG, van OostenAL, BarrandonO, WrayJ, YamanakaS, ChambersI, ASmith. 2009. Nanog is the gateway to the pluripotent ground state. Cell, 138:722–737.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
