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| The Influence of 5-Azacytidine and Trichostatin A on Expression Efficiency of Exogenous DsRed Gene in Transgenic Cashmere Goats (Capra hircus) |
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Abstract Transgene silencing is a widespread phenomenon in transgenic plants and animals, bringing new problems for the cultivation and application of genetically modified organisms. In this study, we analyzed the expression of exogenous Red Fluorescence Protein from Discosoma sp. (DsRed) gene in 2 healthy transgenic Cashmere goats (Capra hircus) and transgenic nuclear-donor cells were analyzed. The expression of an exogenous DsRed gene in fibroblasts obtained from the ear tip was significantly higher than that in the epidermis cells from transgenic Cashmere goats (P＜0.05). After bisulfite sequencing, the methylation status of CMV promoter in 2 healthy transgenic Cashmere goats fibroblast had been checked. The CMV promoter showed high methylation status in 2 samples: ZK0310-1: 89.09%, ZK110324-1: 86.36%. After treatment with the methylation inhibitor 5-Azacytidine (5-Az), the DsRed expression in transgenic goat cells was significantly increased(P＜0.05). After treatment with the histone deacetylase inhibitor Trichostatin A (TSA) in the concentration range of 0~1 μmol/L of TSA, DsRed mRNA expression quantity and concentration of the TSA showed dose-response relationship(R2=0.985, P＜0.01). The optimal drug concentration of 2 cell type isolated from 2 transgenic Cashmere goats were: ZK0311-1 fibroblast, 5-Az: 5 μmol/L、TSA: 1.0 μmol/L; ZK0311-1 epidermis cells, 5-Az: 20 μmol/L, TSA: 1.0 μmol/L; ZK110324-1 fibroblast, 5-Az: 10 μmol/L, TSA: 1.0 μmol/L; ZK110324-1 epidermis cells 5-Az: 5 μmol/L, TSA: 0.75 μmol/L. Time of 5-Az treatment was 7 d; time of TSA treatment was 24 h. After 5-Az and TSA treatment, the increased expression of cell lines DsRed mRNA also caused obvious improvement of red fluorescent protein expression(P＜0.05). After treatment with the methylation inhibitor 5-Az, and CMV promoter methylation in transgenic goat fibroblast was significantly decreased to ZK0311-1: 78.48%, ZK110324-1: 49.7%(P＜0.05). It was proved that 5-Az had obvious inhibition effect of exogenous gene promoter methylation. These results demonstrated that in adult-derived transgenic cells, TSA and 5-Az treatments are effective measures to improve the efficiency of exogenous gene expression. This study has important reference value on how to improve the exogenous gene silencing in transgenic animal production.
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Received: 09 March 2015
Published: 24 July 2015
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