Effect of POU5F1 Expression Level in Clonal Subpopulations of Bovine Fibroblasts Used as Nuclear Donors for Somatic Cell Nuclear Transfer

Abstract

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments.

Expression of pluripotency‐related genes is highly dependent on trichostatin A‐assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos

“…In contrast to our findings and the results of the study by Lee et al (), Sá et al () have indicated that the quantitative profiles of transcriptional activity for Oct4 ( POU5F1 ) gene in bovine cloned blastocysts were not affected by the RA of analogous mRNA transcripts in the preselected cell strains of ear skin‐derived nuclear donor fibroblasts. However, the multipotency extent, epigenetic plasticity and subsequent reprogrammability of nuclear donor cells are dependent (via positive correlation) not only on the broad spectrum of pluripotency‐related genes, but also on the dynamic homeostasis between their transcriptional activities and epigenomic modification profiles, that is DNA cytosine methylation/demethylation and chromatin histone deacetylation/acetylation levels.…”

Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming

“…SCNT was performed as described 51 , with few modifications. Bovine ovaries were obtained from a local slaughterhouse and transported to the laboratory in saline solution at 38.5 °C.…”

Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming

“…SCNT was performed as described 52 , with few modifications. Bovine ovaries were obtained from a local slaughterhouse and transported to the laboratory in saline solution at 38.5°C.…”