Oct4-enhanced green fluorescent protein transgenic pigs: a new large animal model for reprogramming studies.
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.
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany.
Monika
M
Nowak-Imialek
Wilfried A
WA
Kues
Bjoern
B
Petersen
Andrea
A
Lucas-Hahn
Doris
D
Herrmann
Srividyameena
S
Haridoss
Marianne
M
Oropeza
Erika
E
Lemme
Hans R
HR
Schöler
Joseph W
JW
Carnwath
Heiner
H
Niemann
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000378:metabolism; D002459:Cell Fusion; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005818:Genetic Engineering; D005854:Germ Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells; D007668:Kidney / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics*; D013737:Testis / Q000166:cytology / Q000378:metabolism
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins