Oct4-Enhanced Green Fluorescent Protein Transgenic Pigs: A New Large Animal Model for Reprogramming Studies

Abstract

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.

Toward an ideal animal model to trace donor cell fates after stem cell therapy: Production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene1

“…The heart is one of the organs that exhibits the strongest antofluorescence (Nussbaum et al,, 2007), The greater expression of EGFP from onr EGFP-MSG still enabled us to distinguish the donor cells from the background antofluorescence even in this organ, suggesting the feasibility of tracking our EGFP-MSG-derived cells for various MSG-related therapeutic studies in pig models of human diseases, Transgenic pigs harboring the EGFP gene are a usefnl animal model in regenerative medicine. One of the best examples was demonstrated in a recent publication in which transgenic porcine embryonic fibroblasts harboring the hOGT4-EGFP (also termed OG2) transgene were used as donor cells to make OGá-transgenic pigs by somatic cell nuclear transfer (Nowak-Imialek et al,, 2011), These OGâ-transgenic pigs pave ways to insightful studies on pig germ cell development, as well as reprogramining studies, because any non-EGFP expressing differentiated cells types can be reprogrammed back to the hOGT4-EGFP transgene expressing pluripotent status by fusion with murine embryonic stem cells or by overexpressing the human OCT4 (octamerbinding transcription factor 4), S0X2 [SRY (sex determining region Y)-box 2], KLF4 (Kruppel-like factor 4), and e-MYG (c-Myc proto-oncogene; Nowak-Imialek et al, 2011). Mauy luechauistic studies, iucludiug the detailed epigeuetic regulatory cascade, cau be mouitored in the selected EGFP^ cells duriug the course of the reprogrammiug process.…”

Derivation and Characterization ofSleeping BeautyTransposon-Mediated Porcine Induced Pluripotent Stem Cells

“…The Oct4-EGFP transgenic pigs were produced by SCNT and transferred as reconstructed zygotes to synchronized recipients [41]. One pregnant recipient was sacrificed at day 25 of gestation and fetuses were recovered for fibroblast derivation as described [43].…”

“…The Oct4-EGFP pigs carry the entire 18 kb genomic locus of the murine Oct4, which is one of the best characterized genes involved in pluripotency, controlling expression of an EGFP reporter [38][39][40]. In Oct4-EGFP pigs, the EGFP expression was found to be restricted to pluripotent cells of the germ line [41]. Importantly, EGFP expression was re-activated after experimental reprogramming by either somatic cell nuclear transfer (SCNT), fusion of porcine somatic cells with murine embryonic stem (ES) cells, or viral transduction with a set of 4 reprogramming factors [41].…”

“…In Oct4-EGFP pigs, the EGFP expression was found to be restricted to pluripotent cells of the germ line [41]. Importantly, EGFP expression was re-activated after experimental reprogramming by either somatic cell nuclear transfer (SCNT), fusion of porcine somatic cells with murine embryonic stem (ES) cells, or viral transduction with a set of 4 reprogramming factors [41]. Here, we employed porcine Oct4-EGFP transgenic fibroblasts and a SB transposon-based reprogramming approach to generate pluripotent iPS cells.…”

The Small Molecule Inhibitors PD0325091 and CHIR99021 Reduce Expression of Pluripotency-Related Genes in Putative Porcine Induced Pluripotent Stem Cells

“…pFFs that carry the OCT4-EGFP reporter (Nowak-Imialek et al, 2011) were transfected with Sleeping Beauty transposon plasmids containing the cDNA sequences for porcine OCT4, SOX2, C-MYC, KLF4 (SB-CAG-pOSMK-ires-Tomato), as well as porcine NANOG and human LIN28 (SB-Ef1a-pNA-NOG-ires-hLIN28), together with a plasmid coding for the Sleeping Beauty transposase (SBx100). The expression vectors and the transfection procedure were described earlier .…”