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Review
. 2016 Jun;25(3):289-306.
doi: 10.1007/s11248-016-9929-5. Epub 2016 Feb 19.

Pluripotent Stem Cells and Livestock Genetic Engineering

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Free PMC article
Review

Pluripotent Stem Cells and Livestock Genetic Engineering

Delia A Soto et al. Transgenic Res. .
Free PMC article

Abstract

The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs.

Keywords: Embryonic stem cells; Gene editing; Livestock; Pluripotency; Transgenic animals.

Figures

Fig. 1
Fig. 1
ESCs and EpiSCs exist in two temporally distinct developmental states. ESCs represent the epiblast of pre-implantation mouse embryos, they can re-enter embryogenesis at any developmental stage, their pluripotency relies on LIF and 2i culture systems, a distal enhancer (DE) drives Oct4 expression, and both X-chromosomes are in an active state in female cells. EpiSCs instead, are derived from post-implantation embryos existing in a more developmentally advanced state. EpiSCs are incapable of re-entering embryogenesis at a pre-implantation stage, their pluripotency relies on FGF2/Activin signaling, a proximal enhancer (PE) drives Oct4 expression, and one of the X-chromosomes in female cells is inactive. Beside these differences, both cell types express core transcription factors OSN (Oct4, Sox2, and Nanog)
Fig. 2
Fig. 2
Approaches for generating transgenic animals from PSCs. Edited PSCs can be used to create transgenic animals by microinjection into a normal host embryo, a host embryo incapable of developing its own germline or a tetraploid host embryo. Respectively, each of these techniques increases the contribution of PSCs to the animal's germline. Edited PSCs can also contribute their nuclei for cloning by nuclear transplantation or potentially be used to generate in vitro functional gametes. The greatest advantage of these methodologies is that homozygous mutants can be produced in just one generation, largely reducing the number of animals and time needed to produce mutant founders

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