Abstract
Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere‐like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.
Highlights
For two decades, since the creation of the cloned first lamb using a somatic cell donor, the scientific community has strived to improve the efficiency of somatic cell nuclear transfer (SCNT) in mammals (Campbell, McWhir, Ritchie, & Wilmut, 1996; Gábor, 2018)
Proper nuclear remodeling and epigenetic reprogramming of donor cells is revered to be the key to improving SCNT and is thoroughly under examination
Improper nuclear reprogramming has been attributed to the developmental defects which are sometimes observed in clones; the genome of somatic cells must be reset to express genes consistent with appropriate progression of embryonic development
Summary
Since the creation of the cloned first lamb using a somatic cell donor, the scientific community has strived to improve the efficiency of somatic cell nuclear transfer (SCNT) in mammals (Campbell, McWhir, Ritchie, & Wilmut, 1996; Gábor, 2018). While other emerging techniques are improving, such as zygote injection of CRISPR/ Cas, SCNT is still one of the most efficient ways to reliably create genetically engineered animals. A major hurdle for cow and pig iPSCs is that both 1) require continued expression of the introduced transgenes to maintain some degree of pluripotency, 2) have limited passage life, and 3) are not able to form teratomas in immunodeficient mice (reviewed in Ezashi, Yuan, & Roberts, 2016). Due to these challenges, SCNT is still popular in swine
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