Abstract
Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by in vitro differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes.
Highlights
Over the last three decades, the establishment of pluripotent cell lines from preimplantation mouse embryos has been considered to be one of the biggest events in developmental biology [1,2]
Epiblast stem cell (EpiSC)-like porcine embryonic stem cells (pESCs) were cultured in porcine embryonic stem cell media (PESM)
In vitro produced blastocysts were used for the derivation of EpiSC-like pESCs as previously described [8,37]
Summary
Over the last three decades, the establishment of pluripotent cell lines from preimplantation mouse embryos has been considered to be one of the biggest events in developmental biology [1,2]. These cells, known as embryonic stem cells, have in vivo and in vitro differentiation potentials into three germ layers and can proliferate infinitely. Mouse epiblast stem cells (EpiSCs) and induced pluripotent stem cells (iPSCs) were derived from postimplantation embryos and somatic cells, respectively [3,4]. In nonpermissive lines such as human and pig, cells are only derived into the ‘‘primed’’ state, such as epiblast stem cells, if no additional treatment such as genetic manipulation and chemicals are performed [6,7,8]
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