Generation of induced pluripotent stem cells from domestic goats

Abstract

The creation of genetically modified goats provides a powerful approach for improving animal health, enhancing production traits, animal pharming, and for ensuring food safety all of which are high-priority goals for animal agriculture. The availability of goat embryonic stem cells (ESCs) that are characteristically immortal in culture would be of enormous benefit for developing genetically modified animals. As an alternative to long-sought goat ESCs, we generated induced pluripotent stem cells (iPSC) by forced expression of bovine POU5F1, SOX2, MYC, KLF4, LIN-28, and NANOG reprogramming factors in combination with a MIR302/367 cluster, delivered by lentiviral vectors. In order to minimize integrations, the reprogramming factor coding sequences were assembled with porcine teschovirus-1 2A (P2A) self-cleaving peptides that allowed for tri-cistronic expression from each vector. The lentiviral-transduced cells were cultured on irradiated mouse feeder cells in a semi-defined, serum-free medium containing fibroblast growth factor (FGF) and/or leukemia inhibitory factor (LIF). The resulting goat iPSC exhibit cell and colony morphology typical of human and mouse ESCs-that is, well-defined borders, a high nuclear-to-cytoplasmic ratio, a short cell-cycle interval, alkaline phosphatase expression, and the ability to generate teratomas in vivo. Additionally, these goat iPSC demonstrated the ability to differentiate into directed lineages in vitro. These results constitute the first steps in establishing integration and footprint-free iPSC from ruminants. Mol. Reprod. Dev. 82: 709-721, 2015. © 2015 Wiley Periodicals, Inc.

Chick derived induced pluripotent stem cells by the poly‐cistronic transposon with enhanced transcriptional activity

“…It has also been reported that Lin28 inhibits the reversion of reprogrammed cells (Takahashi et al, ; Tanabe et al, ). Hence, this six‐factor approach was found to be beneficial for improving the efficiency of establishment of chick iPS cells, as well as in case of unknown mammalian species (Sandmaier et al, ; Tomioka et al, ). Moreover, we also observed that reprogramming efficiency in swine derived fibroblasts was much lower than that of mice or human (Fukuda et al, ).…”

“…It has also been reported that Lin28 inhibits the reversion of reprogrammed cells (Takahashi et al, 2014;Tanabe et al, 2013). Hence, this six-factor approach was found to be beneficial for improving the efficiency of establishment of chick iPS cells, as well as in case of unknown | 999 mammalian species (Sandmaier et al, 2015;Tomioka et al, 2010).…”

The modification of mitochondrial energy metabolism and histone of goat somatic cells under small molecules compounds induction

“…Then Alkaline phosphatase activity was detected using Alkaline Phosphatase Detection Kit (Millipore, 2617338) according to the manufacturer’s instructions. For immunofluorescence, the operational procedure could refer to Sandmaier’s procedure (Sandmaier et al, ), the primary antibodies included OCT4 (1:300, Abcam, ab18976), SOX2 (1:300, Abcam, ab97959) and the appropriate secondary antibody (anti‐Rabbit IgG H&L (Alexa Fluor® 488), 1:200, Abcam, ab150073) were used for 1 hr in the dark. Nuclei were counterstained with Hoechst33342.…”

Genome editing for disease resistance in livestock

“…Alternatively, small ruminants have also been generated by SCNT (25)(26)(27) and goats by sperm-mediated gene transfer (SMGT) (28). The generation of chimeric embryos from (non-edited) iPSCs has been demonstrated in sheep (29), whilst in goats the generation of iPSCs has been reported, no editing or chimeric integration has been demonstrated yet (30,31).…”