Scriptaid Improves Cashmere Goat Embryo Reprogramming by Affecting Donor Cell Pluripotency Molecule NANOG Expression

Abstract

Currently, the efficiency of somatic cell nuclear transfer (SCNT) technology is relatively low, primarily owing to reprogramming abnormalities in donor cells or reconstructed embryos. Using histone deacetylase inhibitor (HDACi) to artificially alter the epigenetic modifications of donor cells and improve the reprogramming ability of reconstructed embryos is effective in improving nuclear transfer efficiency. In this study, we used Albas cashmere goat cells as donor cells, treated them with Scriptaid, and constructed embryos using SCNT. The results suggest that donor cell treatment with Scriptaid significantly increased the cellular histone acetylation modification level, perturbed the expression of the pluripotency molecule NANOG, altered the reprogramming ability of embryos, and increased the developmental rate of SCNT-reconstructed embryos. Scriptaid inhibited donor cell proliferation, induced apoptosis, and blocked the G0/G1 phase of the cell cycle. These results provide a new research direction for improving SCNT efficiency and a new perspective in the fields of regenerative medicine, agriculture, and animal husbandry.

Keywords: NANOG; SCNT; Scriptaid; cashmere goat; histone acetylation.

Figure 1

The effect of Scriptaid on cell growth activity and proliferation. (A) CCK8 assay for the effect of Scriptaid on the growth activity of gFFCs, with drug concentration in the horizontal coordinate and cell viability in the vertical coordinate. (B) CCK8 assay for the effect of Scriptaid on the growth activity of gADSCs. (C) CCK8 assay for the effect of Scriptaid on the growth activity of gMDSCs. (D) Statistical analysis of proliferation and cell proliferation rate of gFFCs by EdU assay. (E) Statistical analysis of proliferation and cell proliferation rate of gADSCs by EdU assay. (F) Statistical analysis of cell proliferation and cell proliferation rate of gMDSCs detected by EdU. Data (n ≥ 3) are represented as the mean ± SD. * p < 0.1, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 2

The effect of Scriptaid on histone acetylation levels in nuclear donor cells. (A) WB of Kac protein expression in gFFCs and grayscale analysis. (B) WB of Kac protein expression in gADSCs and grayscale analysis. (C) WB of Kac protein expression in gMDSCs and grayscale analysis. (D) mRNA level expression of HATs and HDACs in gFFCs. (E) mRNA level expression of HATs and HDACs in gADSCs. (F) mRNA level expression of HATs and HDACs in gMDSCs. (G) WB of histone acetylation site expression and grayscale analysis in gFFCs. (H) WB of protein acetylation site expression in gADSCs group and grayscale analysis. (I) WB of protein acetylation site expression in gMDSCs group and grayscale analysis. Data (n ≥ 3) are represented as the mean ± SD. ns > 0.1, * p < 0.1, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 3

Effects of Scriptaid on pluripotency genes in nuclear donor cells. (A) qRT-PCR detection of mRNA expression level of pluripotency-related genes in gFFCs. (B) qRT-PCR detection of mRNA expression level of pluripotency-related genes in gADSCs. (C) qRT-PCR detection of mRNA expression level of pluripotency-related genes in gMDSCs. (D) WB detection of NANOG protein expression level and grayscale analysis. Data (n ≥ 3) are represented as the mean ± SD. ns > 0.1, ** p < 0.01 and **** p < 0.0001.

Figure 4

Effect of Scriptaid on apoptosis in donor cells. (A–C) Flow cytometry of Annexin V-FITC/PI staining to detect the apoptosis rate of gFFCs, gADSCs, and gMDSCs. (D–F) Western blot detection of apoptosis-related protein expression of gFFCs, gADSCs, and gMDSCs and grayscale analysis. (G–I) qRT-PCR to detect the expression of mRNA level of apoptosis-related genes in gFFCs, gADSCs and gMDSCs. Data (n ≥ 3) are represented as the mean ± SD. ns > 0.1, * p < 0.1, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 5

Effects of Scriptaid on the donor cell cycle. (A) Flow cytometry to detect the cell cycle of gFFCs, gADSCs, gMDSCs and statistical analysis of each period. (B–D) qRT-PCR to detect the expression of cycle-related gene mRNA levels in gFFCs, gADSCs, and gMDSCs. Data (n ≥ 3) are represented as the mean ± SD. ns > 0.1, * p < 0.1, ** p < 0.01 and **** p < 0.0001.

Figure 6

The SCNT reconstructed embryo development map.