ABSTRACT
The purpose of this study was to investigate the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on transgene expression and development of porcine transgenic cloned embryos, specifically focusing on effects derived from TSA-treated donor cells or TSA-treated reconstructed embryos. The results showed that TSA treatment on reconstructed embryos modified the acetylation status, which significantly improved the development of porcine somatic cell nuclear transfer (SCNT) embryos in vitro, but not donor cells. Furthermore, the treatment of reconstructed embryos with TSA enhanced expression of the pluripotency-related gene POU5F1 and stimulated expression of the anti-apoptotic gene BCL-2. Enhanced green fluorescent protein (EGFP) mRNA expression of every group dropped drastically from donor cells to blastocysts. Interestingly, TSA is likely to prevent a decline in EGFP expression in nuclear reprogramming of porcine SCNT embryos. However DNA hypomethylation induced by modified histone acetylation of donor cells treated with TSA was significantly more effective in increasing EGFP expression in SCNT blastocysts. In conclusion, the acetylation status of both donor cells and reconstructed embryos modified by TSA treatment increased transgene expression and improved nuclear reprogramming and the developmental potential of porcine transgenic SCNT embryos.
Subject(s)
Blastocyst/metabolism , Embryonic Development/drug effects , Histones/metabolism , Hydroxamic Acids/pharmacology , Nuclear Transfer Techniques/veterinary , Acetylation , Animals , Animals, Genetically Modified , Cloning, Organism , DNA Methylation , Embryo Culture Techniques , Green Fluorescent Proteins/metabolism , Histone Deacetylase Inhibitors , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , SwineABSTRACT
The purpose of this study was to enhance the production of transgenic cloned embryos with porcine reproductive and respiratory syndrome virus (PRRSV) shRNA expression cassettes. To construct transgenic vector with expression targeting against PRRSV, PRRSV shRNA expression cassettes were inserted into pEGFP-N1 and the ability of resulting recombinant plasmid pEGFP-G1 inhibiting virus replication was examined in Marc-145 cells. Results showed that PRRSV replication could be significantly inhibited by pEGFP-G1 in Marc-145 cells compared with the control. The pEGFP-G1 plasmid was used to deliver a transgene expressing EGFP and the PRRSV shRNA into porcine fetal fibroblasts (PFF). Fluorescent-positive cells were used as nuclear donors for somatic cell nuclear transfer (SCNT) to produce shRNA-EGFP transgenic cloned embryos. The effects of trichostatin A (TSA) on production of transgenic cloned embryos were investigated. Reconstructed embryos were designed into 4 groups: Donor cells of Group A were treated with 50nM TSA for 24h before SCNT. Reconstructed embryos of Group B were treated with 50nM TSA for 24h after activation. Both donor cells and reconstructed embryos in Group C were treated with TSA and Group D were the control without TSA treatment. The results showed no difference (p>0.05) in cleavage rates among the 4 groups; however, blastocyst developmental rates of Group B and C (30.9% and 42.0%, respectively) were higher than for Group A and D (21.2% and 22.1%, respectively) with Group C highest among groups (p<0.05). Interestingly, EGFP expression intensity of transgenic cloned blastocysts of Group A was the highest. Our results provide promising evidence toward a new approach for production of transgenic cloned pigs with resistance to PRRSV and possibly a wide variety of other porcine diseases.
