| Varied nontransgenic and transgenic mammals have been cloned successfully by somatic cell nuclear transfer(SCNT). However, the efficiency of nuclear transfer is still very low regardless of the species. Recent studies show that epigenetic reprogramming plays a central role in the development of cloned embryos generated by SCNT, and it is believed that aberrant reprogramming leads to the abnormal development of most cloned embryos. It has been demonstrated that the developmental competence of SCNT embryos in several species were significantly enhanced via treatment of histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). The aim of our present study was to establish the optimal TSA treatment in order to improve the development of cloned porcine embryos and examine the effect of TSA on their development and expression of transgenic GFP. Results we found were as follows:Cloned reconstructed embryos from porcine fetal fibroblast cells were treated with different concentrations of TSA (0, 20, 40, 60, 80, 100nM, respectively) for 12 h following oocyte activation. At 7d after oocyte activation, more TSA-treated cloned embryos developed to blastocyst stage with 40 nM-treated clones showing the highest score, 35.65±9.58% compared with 13.00±3.02% of control embryos(P<0.05). As 40 nM TSA-treated cloned embryos developed to 4-cell stage increased remarkably in comparison with the control(P<0.05), our data indicate that 40nM TSA-treatment after SCNT in porcine can dramatically facilitate the happening of zygotic gene activation(ZGA) occurred at 4-cell stage. Treated reconstructed embryos with 40nM TSA for 12 h and 24h, no significant diference of the blastocyst rate were observed between the periods with the control(13.24±5.55%), 16.32±1.51% and 14.93±4.91% respectively(P>0.05). These results demonstrated that the in vitro development potency of cloned porcine embryos were enhanced both by 40nM TSA treated 12h or 24h.Reconstructed embryos from porcine bone marrow mesenchymal stem cells(PMSCs), porcine fetal fibroblast cells(PFFs) and cumulus cells were treated with 40nM TSA for 12 h following oocyte activation. No significant diference of the cleavage and blastocyst rates were observed between three treatment groups(68.73±14.32%, 64.30±15.07%, 69.56±12.41% and 17.39±14.64%, 12.65±4.82%, 2.90±5.02%, respectively; P>0.05). Cloned embryos reconstructed with porcine eGFP-transgenic fetal fibroblast cells were treated with different concentrations of TSA (0, 20, 40, 60, 80, 100, 120 and 140nM, respectively) for 12 h following oocyte activation. No significant diference of the blastocyst rate were observed between treatment groups(P>0.05). These results shown that TSA can enhance the in vitro development potency of cloned porcine embryos reconstructed with different donor cells.Blastocyst rate of human-porcine interspecies nuclear transfer(hp-ISNT) embryos reconstructed with human foreskin fibroblast cells and porcine ooplasmics were observed significantly lower than parthenogenetic group(1.07±1.27% and 21.06±5.92%, respectively; P<0.05). As 4-cell embryos of hp-ISNT were reported significantly lower than parthenogenetic group(62.84±18.57% and 87.13±4.42%, respectively; P<0.05), it has been demonstrated that lower developmental rate of hp-ISNT embryos concerned possibly with incomplete happening of zygotic gene activation(ZGA) occurred at 4-cell stage. Treated reconstructed hp-ISNT embryos with 40nM TSA for 24h, there was no difference regarding invitro development rate among groups(P>0.05). These results shown that inability of TSA enhanced notably the in vitro development potency of hp-ISNT embryos.Blastocyst rate of porcine reconstructed embryos from eGFP-transgenic fetal fibroblast cells treated with 5nM TSA for 24h were observed as 7.84±5.31%, reducing significantly with the control in which both reconstructed embryos and their doner cells were not treated with TSA(P<0.05). Further, eGFP-transgenic fetal fibroblast cells treated with 5nM TSA for 24h were used as donor cells, from which porcine reconstructed embryos were treated with 40nM TSA for 12h; the blastocyst rate of porcine SCNT embryos were detected as 48.96±10.17%, significantly larger than the control group(P<0.05). These results indicated that the in vitro development potency of transgenic NT embryos were obviously improved via treatment of TSA.We tested the expression of the transgenic enhancer green fluorescence protein (eGFP) gene in cloned preimplantation embryos by fluorescence detection. eGFP-derived fluorescence was observed obviously in the nuclears of 1-cell reconstructed embryos after 2h and 12h activated. However, eGFP expression detected in embryos at the 2～4-cell stages was lower than the 1-cell embryos. Furthermore, the fluorescence intensity in the nuclears of blastocysts were stronger than the 4-cell embryos.Using reverse transcription-polymerase chain reaction(RT-PCR), here we compared the expression patterns of eGFP genes in reconstructed embryos treated with and without TSA. The mRNA level of eGFP gene indicated in reconstructed embryos, inwhich only donor cells were treated with 5nM TSA for 24h(G2 group), were significantly higher than the control(G1 group) (P<0.05). But the expression patterns of eGFP gene exhibited similarly in the two groups, showing that the mRNA level of eGFP gene increased gradually with the development of NT embryos. While both reconstructed embryos and donor cells were treated with TSA(G3 group), the eGFP mRNA level of reconstructed embryos from the G3 group shown no significant difference with the G2 group at the 1-cell stage(P>0.05). With the development of cloned embryos, the eGFP mRNA level of reconstructed embryos in the G3 group was decreased gradually as the level of G1 group. It has been demonstrated that expression level of transgenic eGFP gene were influenced by the treatment of TSA.
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