Treatment of Donor Cells and Reconstructed Embryos with a Combination of Trichostatin-A and 5-aza-2'-Deoxycytidine Improves the Developmental Competence and Quality of Buffalo Embryos Produced by Handmade Cloning and Alters Their Epigenetic Status and Gene Expression.

摘要: The application of cloning technology on a large scale is limited by very low offspring rate primarily due to aberrant or incomplete epigenetic reprogramming. Trichostatin A (TSA), histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor DNA methyltransferases, are widely used for altering the status cloned embryos. We optimized doses these modifiers production buffalo embryos handmade examined whether combined treatment with offered any advantage over individual modifier. Irrespective donor cells reconstructed both were treated 50 nM TSA +7.5 nM 5-aza-dC, (1) blastocyst was significantly higher (71.6 ± 3.5, 68.3 ± 2.6, 71.8 ± 2.4, respectively, vs. 43.1 ± 3.4 controls, p < 0.05); (2) apoptotic index lower (5.4 ± 1.1, 9.5 ± 1.0, 7.4 ± 1.3, 19.5 ± 2.1 p < 0.05) similar that in vitro fertilization blastocysts (6.0 ± 0.8); (3) global level H3K18ac (p < 0.01) H3K27me3 (p < 0.05) than controls among all groups; (4) expression epigenetic-(HDAC1, DNMT1, DNMT3a), pluripotency-(OCT4 NANOG), development-related (FGF4) genes, but not SOX2 CDX2, groups. These results demonstrate levels beneficial effects can be obtained following either combination +5-aza-dC. Therefore, there no treating when 5-aza-dC used.