Document Type : Original Article
Cellular and Molecular Biology Research Center, Faculty of Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran;Department of Cell Biology and Anatomical Science, Faculty of Medicine, Shahid Beheshti Univer
Cellular and Molecular Biology Research Center, Faculty of Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran;4Department of Biothechnology, Faculty of Medicine, Shahid Beheshti University of Medical Scie
5Department of Immunology, Faculty of Medicine, Tehran, Iran
Department of Cell Biology and Anatomical Science, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6Department of Molecular and Cellular Biology, University of Guelph, Canada
7Department of Transgenic Animal Science, Stem Cell Technology Research Center, Tehran, Iran
Department of Embryology, Mehr Infertility Institute, Rasht, Iran
Nuclear transfer-embryonic stem cells (NT-ESCs) are genetically identical to the donor’s cells; provide a renewable source of tissue for replacement, and therefore, decrease the risk of immune rejection. Trichostatin A (TSA) as a histone deacetylase in- hibitor (HDACi) plays an important role in the reorganization of the genome and epigenetic changes. In this study, we examined whether TSA treatment after somatic cell nuclear transfer (SCNT) can improve the developmental rate of embryos and establishment rate of NT-ESCs line, as well as whether TSA treatment can improve histone modification in NT-ESCs lines.
Materials and Methods
In this experimental study, mature oocytes were recovered from BDF1 [C57BL/6×DBA/2) F 1 mice] mice and enucleated by micromanipulator. Cumulus cells were injected into enucleated oocytes as donor. Reconstructed embryos were ac- tivated in the presence or absence of TSA and cultured for 5 days. Blastocysts were transferred on inactive mouse embryonic fibroblasts (MEF), so ESCs lines were estab- lished. ESCs markers were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Histone modifications were analyzed by enzyme linked immunosorbent assay (ELISA).
Result of this study showed that TSA treatment after SCNT can improve devel- opmental rate of embryos (21.12 ± 3.56 vs. 8.08 ± 7.92), as well as establishment rate of NT-ESCs line (25 vs. 12.5). We established 6 NT-ESCs in two experimental groups, and three embryonic stem cells (ESCs) lines as control group. TSA treatment has no effect in H3K4 acetylation and H3K9 tri-methylation in ESCs.
TSA plays a key role in the developmental rate of embryos, establishment rate of ESC lines after SCNT, and regulation of histone modification in NT-ESCs, in a man- ner similar to that of ESCs established from normal blastocysts.